CN114026098A

CN114026098A - Benzopyran based compounds, methods and uses thereof

Info

Publication number: CN114026098A
Application number: CN202080047724.5A
Authority: CN
Inventors: 玛尔塔·西尔维娅·弗雷塔斯·达·科斯塔; 马里亚·德·法蒂玛·蒙吉尼奥·巴尔塔萨; 玛丽亚·费尔南达·德·热苏斯·雷戈·派瓦·普罗恩萨; 帕特里夏·马西埃尔; 奥利维娅·亚历山德拉·埃利亚斯·庞特斯
Original assignee: Universidade do Minho
Current assignee: Universidade do Minho
Priority date: 2019-06-28
Filing date: 2020-06-29
Publication date: 2022-02-08
Also published as: EP3990118A1; JP2022539146A; AU2020307678A1; US20240092793A1; WO2020261242A1

Abstract

The present invention relates to a compound or a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph for use in medicine, comprising the following formula (I): wherein R is¹、R²And R³Selecting independently of each other; r¹Selected from aryl or heterocycle; r²Selected from H, alkyl, aryl, alkoxy, halogen, hydroxy, amine, carbonyl, or heterocycle; r³Selected from H or (II).

Description

Benzopyran based compounds, methods and uses thereof

Technical Field

The present invention relates to a novel class of benzopyran-based anti-cancer compounds. The present invention describes the anticancer properties of a new class of benzopyran-based anticancer compounds. The present invention also describes a method for the synthesis and isolation of a novel class of benzopyran-based anticancer compounds.

Background

In 2018, cancer causes nearly 1000 million deaths worldwide, and 1810 thousands of new cancer cases were detected in the same year.¹The incidence of this disease is expected to exceed 4300 ten thousand cases after 5 years. This demonstrates the urgency to find new and better treatments. This problem must be addressed worldwide due to the overall increase in the number of cases and associated mortality. In 2018, breast cancer accounted for 6.6% of cancer deaths. Breast cancer is the fifth lethal cancer type; in 2018, 11.6% of new cases (more than 200 ten thousand cases) were detected.²This is the second most common type of cancer in terms of morbidity. The type of treatment is always based on the stage of the cancer, but mainly involves surgery, radiation, conventional chemotherapy, immunizationEpidemic therapy or molecular therapy if molecular markers such as ER, PR and HER-2 are detected. However, with Triple Negative Breast Cancer (TNBC), which is a very aggressive subtype of breast cancer that accounts for approximately 15% -20% of all breast cancers, conventional therapies have limited efficacy and no molecular or personalized therapies have been developed. Due to this poor prognosis, high mortality is associated with this breast cancer subtype.³

The benzopyranyl backbone has inspired medicinal chemists because these compounds occur widely in nature and possess a variety of biological properties.⁴In recent years, research has gained attention regarding the synthetic and biological importance of such naturally occurring scaffolds and synthetic derivatives thereof.

The Costa et al reference "Tetrahedron" describes a one-pot cascade involving salicylaldehyde and an aryleneaminoacetonitrile for the preparation of 2-aryl-1, 9-dihydrobenzopyrano [3,2-d ] imidazoles. This document further describes a novel fused tricyclic system combining benzopyran and imidazole as an alternative drug candidate with improved pharmacological properties. However, the described system does not allow the production of molecules in which the aromatic units incorporate different substituents.

These facts are disclosed to show the technical problems solved by the present invention.

Disclosure of Invention

In particular, the present invention describes methods for the synthesis of novel benzopyranyl compounds by binding at least two different molecules, thereby producing a compound having the relevant biological property of interest.

The synthesis of benzopyranyl compounds described in the prior art does not allow the isolation of these novel compounds. The methods described in the present invention allow the synthesis and isolation of novel benzopyranyl compounds that are surprisingly effective when used as anti-cancer compounds in several cancer subtypes, such as breast cancer, renal cell carcinoma, acute leukemia and glioma. The compounds are particularly effective against breast and renal cell carcinomas.

In one embodiment, the synthesized compounds are tested for their anti-cancer potential by in vitro screening using appropriate cell lines. The activity of the compounds was studied at various levels, including the effects on cell viability, proliferation, migration, invasion, cell death and metabolism.

In one embodiment, the toxic effects of benzopyranyl compounds were also tested in non-neoplastic cell lines.

In one embodiment, the Caenorhabditis elegans (c. elegans nematode) model is used for early in vivo toxicity screening of benzopyranyl compounds. In vivo toxicity was also evaluated using mice as a model.

In one embodiment, the anticancer properties of benzopyran based compounds have also been identified using an in vivo CAM (chicken chorioallantoic membrane) assay that assesses the efficacy and angiogenesis of the novel benzopyrans. In vivo efficacy was further evaluated using mice as a model.

The compounds that are the subject of the present invention exhibit unique and promising anti-cancer properties.

One aspect of the present invention pertains to a compound comprising the following formula or a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof:

wherein

R¹、R²And R³Selecting independently of each other;

R¹selected from H, alkyl, aryl, alkoxy, acyl, halogen, nitro, hydroxyl, amine, acylAmines, ketones, esters, heterocycles;

R²selected from: H. alkyl, aryl, alkoxy, acyl, halogen, nitro, hydroxy, amine, amide, carbonyl, ketone, ester, heterocycle;

R³selected from H, alkyl, aryl, alkoxy, acyl, halogen, nitro, hydroxy, amine, amide, ketone, ester, heterocycle or

Another aspect of the invention relates to a compound comprising the formula or a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof:

R¹selected from aryl or heterocycle;

R²selected from: H. alkyl, aryl, alkoxy, halogen, hydroxy, amine, carbonyl, or heterocycle;

R³is selected from H or

Preferably provided that it does not comprise

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph, wherein: r¹Selected as aryl; and R is²Selected from H, alkyl, alkoxy, halogen, hydroxy or amine.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph, wherein the dimer is preferably a homodimer.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein R is a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof¹Is a substituted aryl group.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein R is a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof¹Selected from the following list: hydroxyphenyl, hydroxy-methoxyphenyl, hydroxy-bromophenyl, hydroxy-chlorophenyl, fluorophenyl, bromophenyl, 24-chlorophenyl, phenyl, methoxyphenyl, difluoro-hydroxyphenyl, ethoxyphenyl or bromo-hydroxy-methoxyphenyl.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein R is a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof¹Is 2-hydroxyphenyl, 2-hydroxy-3-methoxyphenyl, 2-hydroxy-5-bromophenyl, 2-hydroxy-5-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-chlorophenyl, phenyl, 3-hydroxyphenyl, 2-methoxyphenyl, 3, 5-difluoro-2-hydroxyphenyl, 4-ethoxyphenyl or 2-bromo-3-hydroxy-4-methoxyphenyl.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein R is a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof²Is a substituted or unsubstituted aryl group.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereofForm (I) wherein R²Selected from the following list: H. 5-methoxy, 7-bromo, 7-chloro or 7-fluoro.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein R is a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof³Is a benzopyran unit or H.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein R is a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof³Selected from the following list: H. 2- (4-fluorophenyl) -5-methoxy-1, 9-dihydrobenzopyrano [2,3-d]Imidazole unit, 2- (4-bromophenyl) -5-methoxy-1, 9-dihydrobenzopyrano [2,3-d]Imidazole unit or 2- (2-fluorophenyl) -5-methoxy-1, 9-dihydrobenzopyrano [2,3-d]An imidazole unit.

In one embodiment, the compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein the compound is selected from the following list:

in one aspect of the invention, the disclosed compounds are used in medicine or veterinary medicine.

In one aspect of the invention, the compounds may be used in the treatment, therapy or diagnosis of diseases characterized by benign or malignant cell proliferation or by areas of neovascularization or excessive vascularization, or cancer.

In one aspect of the invention, the compounds may be used in the treatment, therapy or diagnosis of hyperproliferative tissue or neoplasia.

In one aspect of the invention, the compounds may be used in the treatment, therapy or diagnosis of breast cancer, renal cell carcinoma, leukemia, glioma or glioblastoma.

In one aspect of the invention, the compounds may be used in the treatment, therapy or diagnosis of renal cell carcinoma, leukemia, glioma, glioblastoma, breast cancer.

In one aspect of the invention, the compounds may be used in the treatment, therapy or diagnosis of triple negative breast cancer, acute renal cell carcinoma, luminal breast cancer, basal-like breast cancer, acute leukemia.

Another aspect of the invention relates to pharmaceutical compositions comprising at least one of the disclosed compounds.

In one embodiment, the disclosed pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In one embodiment, the disclosed pharmaceutical composition further comprises an antiviral agent, an analgesic, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic agent, an antibiotic, an antifungal agent, an antiparasitic agent, or a diuretic, or mixtures thereof.

In one embodiment, the disclosed pharmaceutical composition further comprises a filler, a binder, a disintegrant, a lubricant, or a mixture thereof.

In one embodiment, the disclosed pharmaceutical composition is for intradermal or transdermal therapy, or local or systemic, or intravenous therapy, or a combination thereof.

Another aspect of the invention relates to a method of obtaining the disclosed compounds, comprising the steps of:

concentrated HCl (1.1 equiv.) was added to 2-imino-8-methoxy-2H-chromen-3-amine (0.150 mg; 0.79mmol) in 1mL CH₃Orange color in CNIn solution;

the mixture was stirred at room temperature (20 ℃) for 10-15 minutes (direct precipitation was observed);

filtering the solid, preferably by simple filtration, to obtain 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride;

aldehyde (1.1-1.7 equiv.) was added to 3-amino-8-methoxy-2H-benzopyran-2-imido chloride (0.25-0.35mmol) in CH₃CN (1-2 mL);

the suspension is stirred at 60 ℃ for 24-48 hours,

the resulting solid is filtered to isolate the pure product, preferably with CH₃CN cleaning;

optionally when in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃(0.05M) of an aqueous solution, filtration and washing with water, resulting in the pure product 2- (5-methoxy-3, 9-dihydrobenzopyrano [2, 3-d)]Imidazole.

In one embodiment, for 5,5' -dimethoxy-2, 2' -diphenyl-1, 1',9,9' -tetrahydro-9, 9' -dibenzopyrano [2,3-d ]]Preparation of imidazole by addition of aldehyde (1-1.2 equiv.) to 2-imino-8-methoxy-2H-benzopyran-3-amine in CH₃CN (1-2mL) and the solution was stirred at 80 ℃ for 7-24 hours. The solid product began to precipitate slowly, was filtered and washed with CH₃CN washed and identified as pure product 5,5' -dimethoxy-2, 2' -diphenyl-1, 1',9,9' -tetrahydro-9, 9' -dibenzopyrano [2,3-d ]]Imidazole.

Another aspect of the invention relates to nanoparticles comprising the disclosed compounds and/or the disclosed pharmaceutical compositions.

In one embodiment, nanoparticles comprising the disclosed compounds and/or pharmaceutical compositions are encapsulated by nanoparticles.

Another aspect of the invention relates to kits comprising the compounds and/or the disclosed pharmaceutical compositions.

Another aspect of the invention relates to the use of the compound or a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph for medical or veterinary use. In particular for use in the treatment or therapy of diseases characterised by benign or malignant cell proliferation or by areas of neovascularisation or cancer. In particular for use in the treatment, therapy or diagnosis of hyperproliferative tissue, such as that associated with cancer. In addition, for use in the treatment, therapy or diagnosis of breast cancer, renal cell carcinoma, acute leukemia and glioma. Furthermore, use for the treatment, therapy or diagnosis of glioblastoma and triple negative breast cancer.

Based on the definition of the International Union of Pure and Applied Chemistry (IUPAC), an alkyl group is defined as a monovalent group-C-derived from an alkane by the removal of a hydrogen atom from any carbon atom_nH_2n+1. Groups derived by removal of a hydrogen atom from a terminal carbon atom of an unbranched alkane form the n-alkyl (n-alkyl) subclass H (CH)₂)_n. Group RCH₂、R₂CH (R.noteq.H) and R₃C (R.noteq.H) is a primary, secondary and tertiary alkyl group, respectively. Aryl groups are derived from aromatic hydrocarbons (both monocyclic and polycyclic) by removing a hydrogen atom from a ring carbon atom.

"alkyl" includes "lower alkyl" and extends to cover carbon fragments having up to 30 carbon atoms. Examples of alkyl groups include octyl, nonyl, norbornyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, eicosyl, 3, 7-diethyl-2, 2-dimethyl-4-propylnonyl, 2- (cyclododecyl) ethyl, adamantyl, and the like.

"lower alkyl" means an alkyl group having 1 to 7 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 2-methylcyclopropyl, cyclopropylmethyl, and the like.

In the present invention, halogen means an element selected from the following list: fluorine (F), chlorine (Cl), bromine (Br), iodine (I), astatine (At).

In the present invention, the term "heterocycle" means a ring in which at least one of the atoms forming the main chain of the ring is not carbon. Unless otherwise specified, a heterocyclic ring may be a saturated, partially unsaturated, or fully unsaturated ring. "saturated heterocycle" refers to a heterocycle containing only single bonds between ring members. "partially saturated heterocycle" refers to a non-aromatic heterocycle containing at least one double bond. The term "heteroaromatic ring" refers to a fully unsaturated aromatic ring in which at least one atom forming the ring backbone is not carbon. Typically, the heteroaromatic ring contains no more than 4 nitrogens, no more than 1 oxygen, and no more than 1 sulfur. Unless otherwise indicated, the heteroaryl ring may be attached through any available carbon or nitrogen by replacement of a hydrogen atom on said carbon or nitrogen. The term "heteroaromatic bicyclic ring system" denotes a ring system consisting of two fused rings wherein at least one of the two rings is a heteroaromatic ring as defined above.

The term "carbocyclic" denotes a ring wherein the atoms forming the ring backbone are selected from carbon only. Unless otherwise specified, carbocycles may be saturated, partially unsaturated, or fully unsaturated rings. When a fully unsaturated carbocyclic ring satisfies the huckel's rule, then the ring is also referred to as an "aromatic ring". "saturated carbocyclic ring" means a ring having a main chain consisting of carbon atoms connected to each other by single bonds; unless otherwise specified, hydrogen atoms occupy the remaining valences of the carbon atoms.

Drawings

The following drawings are provided to illustrate preferred embodiments of the present invention and should not be construed as limiting the scope of the invention.

FIG. 1 shows the effect of benzopyranyl compounds on MCF-7 cell migration as assessed by the wound-healing assay (with individual 1/2IC for each compound)₅₀And IC₅₀Values were processed for 12, 24, 48, 72 hours).

FIG. 2 shows the effect of benzopyranyl compounds on Caki-2 cell line migration as assessed by the wound-healing assay (with respective IC for each compound)₅₀Value treatment for 12, 24, 36, 48 hours).

FIG. 3 shows the effect of benzopyranyl compounds on 786-O cell proliferation after 48 hours of treatment.

FIG. 4 shows the evaluation by annexin V/PI assayFlow cytometric analysis of MCF-7 cell viability. FIG. 4A is IC of compounds or treated with 0.5% DMSO (control)₅₀Representative dot plots of MCF-7 cells treated at concentrations for 12 hours and 24 hours. Fig. 4B shows quantification of the percentage of cells in each quadrant of the dot plot.

Figure 5 shows a flow cytometric analysis of Hs578t cell viability assessed by the annexin V/PI assay. FIG. 5A is IC treated with 0.5% DMSO (control) or with reagents₅₀Representative dot plots of Hs578t cells treated at concentrations for 12 hours and 24 hours. Fig. 5B shows quantification of the percentage of cells in each quadrant of the dot plot.

FIG. 6 shows the use of a corresponding IC₅₀Concentration, immunoblot analysis of total PARP,

caspase

3 and 9, BIM and Bcl-xL after treatment of MCF-7 cells with compounds MC409, MC408, MC406 and MC421 (a) for 24 hours and (B) for 48 hours. Protein levels in lysates were detected by immunoblotting using 12% polyacrylamide gels.

FIG. 7 shows the use of a corresponding IC₅₀Concentration, immunoblot analysis of total PARP,

caspase

3 and 9, BIM, Bcl-xL and Bax after treatment of Hs578t cells with compounds MC409, MC408, MC406 and MC421 (a) for 24 hours and (B) for 48 hours. Protein levels in lysates were detected by immunoblotting using 12% polyacrylamide gels.

FIG. 8 shows flow cytometry analysis of DNA content of MCF-7 cells. FIG. 8A is IC treated with 0.5% DMSO (control) or with reagents₅₀Representative histograms of cell cycle profiles of MCF-7 cells treated at concentrations for 12 and 24 hours. Fig. 8B shows cell quantification in different phases of the cell cycle.

Figure 9 shows flow cytometry analysis of the DNA content of Hs578t cells. FIG. 9A is IC treated with 0.5% DMSO (control) or with reagents₅₀Representative histograms of cell cycle profiles of Hs578t cells treated at concentrations of 12 hours and 24 hours. Fig. 9B shows cell quantification in different phases of the cell cycle.

Figure 10 shows the effect of compounds MC408 and MC421 on the microtubule network of Hs578t (A, B) and MCF-7(C, D) cells.

Fig. 11 shows the strategy used in the determination of toxic effects in c. The insects were cultured in liquid medium for 7 days using heat inactivated bacteria (e.coli) OP50) as food source and in the presence of several concentrations of the compounds MC421, MC406, MC369 and MC408, MC409, MC 407. The rate of food consumption between days 3-5 was used as an indicator of insect development, health and fertility, as after day 3, the insect progeny would also promote a more rapid reduction in food quantity. Examples are shown for hypothetical compound X. DMSO 1% and 5% were used as negative and positive controls for toxicity.

Figure 12 shows that the compounds lack in vivo toxicity in caenorhabditis elegans (c.

FIG. 13 shows the effect of compound MC408 on C57Bl/6 mice.

FIG. 14 shows the results of a series of welfare tests performed on C57BI/6 mice treated with Compound MC 408.

FIG. 15 shows the effect of MC408 treatment on enzymatic liver function in C57Bl/6 mice.

FIG. 16 shows the effect of MC408 treatment on C57Bl/6 mice-trial 2. Fig. 16A is a schematic illustration of an experimental timeline. Fig. 16B shows the body weight of the mice.

Figure 17 shows the in vivo therapeutic effect of MC408 and MC421 on breast cancer Hs578t cell line. Figure 17A shows representative photographs of CAM assays and figure 17B shows percent tumor growth.

FIG. 18 shows flow cytometric analysis of 786-O cell viability assessed by annexin V/PI assay. FIG. 18A is IC treated with 0.5% DMSO (control) or with reagents₅₀Representative dot plots of 786-O cells treated at concentrations for 24 and 48 hours. Fig. 18B shows quantification of the percentage of cells in each quadrant of the dot plot.

FIG. 19 shows the use of a corresponding IC₅₀Concentration, immunoblot analysis of KDM4C, total PARP, Hsp90, total JNK, total p53, Bid, and p21 after 786-O cells treated with compounds MC408 and MC421 (a) for 24 hours and (B) for 48 hours. Cleavage was detected by immunoblotting using 12% polyacrylamide gelProtein level in the hydrolysate.

FIG. 20 shows the phosphorylation forms of total mTOR and mTOR, ERK, VEGFR, and total mTOR after 2 hours of starvation and treatment of 786-O cells with cediranib, compounds MC350, MC415, MC412, MC408, and MC421 at a unique concentration of 2 μ M for 6 hours²Immunoblot analysis of EGFR, AKT, PTEN and AMPK, phosphorylated forms of PDK1, NF-kB and p18 and c-Myc, Hsp90 and PRAS40 proteins. Protein levels in lysates were detected by immunoblotting using a 10% polyacrylamide gel.

FIG. 21 shows the in vivo therapeutic effect of MC408 and MC421 on renal cancer 786-O cell line. Figure 21A shows representative photographs of CAM assays and figure 21B shows percent tumor growth.

FIG. 22 shows flow cytometry analysis of DNA content of 786-O cells. FIG. 22A is IC treated with 0.5% DMSO (control) or with reagents₅₀Representative histograms of cell cycle profiles of 786-O cells treated at concentrations of 24 and 48 hours. Fig. 22B shows cell quantification in different phases of the cell cycle.

FIG. 23 shows 1/2IC with selected compounds₅₀Or IC₅₀Effects of benzopyranyl compounds on a498 tolerance and cell proliferation of parental cell lines after 24 and 48 hours of treatment.

FIG. 24 shows 1/2IC with selected compounds₅₀Or IC₅₀Effects of benzopyranyl compounds on Caki-2 tolerance and cell proliferation of the parental cell line after 24 and 48 hours of treatment.

FIG. 25 shows IC's with rapamycin and cediranib, compounds MC350, MC413, MC408 and MC421₅₀Immunoblot analysis of total ERK and GAPDH and their phosphorylated forms, HK2, LDHA, Hif 2a, MCT1, PKM, PFKL, Hsp90, and c-Myc proteins after a period of 48 hours of treatment of a498 parental (P) and tolerant (R) cells. Protein levels in lysates were detected by immunoblotting using a 10% polyacrylamide gel.

Figure 26 shows the in vivo effect of compounds MC408 and MC421 on angiogenesis. Representative images of in ovo and out ovo CAM assays (20 x magnification) on days 13 and 17.

Figure 27 shows tumor volume and body weight in an orthotopic breast cancer NSG mouse xenograft model.

Detailed Description

In particular, the present invention describes a method for the synthesis of novel benzopyran-based compounds by combining at least two different molecules, thus producing compounds with a relevant biological profile of interest.

In one embodiment, 19 different benzopyran-imidazolyl compounds are isolated. Table 1 shows the benzopyran-based backbone structure and 19 synthesized and isolated benzopyran-imidazolyl compounds.

Table 1: structure of benzopyran compound

In one embodiment, the cytostatic and IC of the synthesized and isolated benzopyran based compounds is determined as part of the evaluation of the antiproliferative activity of the isolated agent₅₀The value is obtained. A complete study of the structure-activity relationship was performed on all the synthesized compounds. Antiproliferative activity against the MCF-7 breast cancer cell line was analyzed. Table 2 shows the IC of the compounds on the breast cancer cell line MCF-7 and on the non-neoplastic cell line MCF-10A₅₀Value (. mu.M) and Selectivity Index (SI). Non-neoplastic cell line MCF-10A was also used to determine combinationSelectivity of the substance for tumor cells. For IC₅₀Compounds with values (concentration required for 50% reduction in cell viability) higher than 20. mu.M, undetermined IC₅₀The value is obtained. To evaluate the cytotoxicity of the test compounds, a Selectivity Index (SI) was calculated.

Table 2: IC of compound on breast cancer cell line MCF-7 and non-neoplastic cell line MCF-10A₅₀Value (. mu.M) and Selectivity Index (SI)

Dimeric compounds provide excellent IC for MCF-7 cell lines₅₀A value which is less than the respective monomer. Monomeric compounds having halogen atoms attached to imidazole moieties in aromatic rings or having no substituents provide smaller ICs₅₀Value (<1 μ M). Having OH or OCH₃/OCH₂CH₃The imidazo-benzopyrans lead to significantly higher ICs₅₀Values, even when halogen atoms are present in the same aromatic moiety.

Promising ICs have also been obtained for some benzopyrans for the non-neoplastic cell line MCF-10A₅₀Values and high SI values were calculated. For dimeric compounds, toxicity to non-neoplastic cells is much lower, resulting in superior SI values (above 60). SI values are generally very promising.

In one embodiment, for comparative studies, monomers and respective dimers were selected and further evaluated in Hs578t and MDA-MB-468 breast cell lines. Table 3 shows the IC of selected benzopyran compounds for breast cancer cell lines Hs578t and MDA-MB-468₅₀Value (. mu.M) and Selectivity Index (SI). These cell lines correspond to the basal cell-like subtype of breast cancer, which is included in the known triple negative subtypes because of their specificity for Estrogen Receptors (ER), Progesterone Receptors (PR) and human epidermal growth factor receptors (EGF)2(HER²) The molecular marker is negative. The clinical manifestations of these breast cancer subtypes are very aggressive and there is still no specific molecular therapy.

Table 3: IC of selected benzopyran Compounds for TNBC cell lines MDA-MB-468, MDA-MB-231 and Hs578t₅₀Value (. mu.M) and Selectivity Index (SI)

Benzopyranyl compounds exhibit very low IC from 0.035 to 0.27 μ M for Hs578t₅₀The value is obtained. Bromine-substituted benzopyranyl Compounds MC408 and dimeric Compounds MC421 show extremely Low IC for Hs578t and MDA-MB 468 cell lines₅₀The value is obtained. The compound MC408 is particularly active (IC)₅₀0.027 μ M). In general, compounds show an even greater interest in anti-proliferative potential for these aggressive breast cancer subtypes and SI values are also excellent.

In one embodiment, the antiproliferative activity of compounds MC408, MC409, MC421 and MC406 was further determined for glioma cell lines (U87, GAMG and GL18) and acute leukemia cell lines (HL-60, KG-1 and Jurkat). This study was conducted to assess whether compounds exhibit the same interesting anti-cancer profile in other cancer cell models. The respective IC was determined by exposing the cells to the respective compounds for 72 hours at the appropriate concentration range₅₀After the values, cell viability was determined using the MTS assay. Table 4 shows the IC of compounds MC408, MC409, MC421 and MC406 on glioma and leukemia cancer cell models₅₀Value (. mu.M).

Table 4: IC of Compounds MC408, MC409, MC421 and MC406 on glioma and leukemia cancer cell models₅₀Value (μ M)

The compounds show excellent antiproliferative capacity due to IC determined for all cell lines₅₀Values are in the nano-molar or low micro-molar range. Compound MC421 showed very high inhibition of cell growth on glioma cell lines, especially for U87 and GAMG. Compound MC408 showed lower antiproliferative ability when compared to other compounds, but IC₅₀The values are still in the low μ M range. All compounds appear to be more selective for the Jurkat cell line in the leukemia model, but superior growth inhibition was also achieved by treatment of other leukemia cell lines with all compounds. These results indicate that these compounds are promising in several cancer cell models and may be considered candidates for cancer therapy.

In one embodiment, the antiproliferative activity of the compounds is further determined for renal cancer cell lines and non-tumor renal cell lines. Table 5 shows the IC's of renal cancer cell lines (786-O, Caki-2, A498) and non-neoplastic cell lines (HK2)₅₀Value (. mu.M) and Selectivity Index (SI). Compounds M955, M1220, M1143 and M1221 do not show any biological activity in the first screen and do not continue IC₅₀And (4) determining. Some compounds show high potency and excellent selectivity index for Renal Cell Carcinoma (RCC) cell lines.

Table 5: IC of renal cancer cell lines (786-O, Caki-2, A498) and non-neoplastic cell lines (HK2)₅₀Value (. mu.M) and Selectivity Index (SI)

In one embodiment, the antiproliferative activity of compounds MC421, MC409, MC408, MC350, MC416, MC410, MC411, MC415, and MC413 against drug resistant renal cancer cell lines is further determined. Table 6 shows the IC's of rapamycin-tolerant A498 and cediranib-tolerant Caki-2 cell line and non-neoplastic cell line (HK2)₅₀Value (. mu.M) and Selectivity Index (SI).

Table 6: IC of rapamycin-tolerant A498 and cediranib-tolerant Caki-2 cell line and nonneoplastic cell line (HK2)₅₀Value (. mu.M) and Selectivity Index (SI).

In one embodiment, the effect of 4 selected compounds (MC408, MC409, MC406 and MC421) on cell migration was further evaluated (fig. 1). Using respective IC₅₀And IC₅₀Half of the value MCF-7 cells were treated for 72 hours and the wound-healing assay was performed. For each compound, IC was used₅₀Half the concentration was evaluated for concentration-dependent effects. FIG. 1 shows the effect of benzopyranyl compounds on MCF-7 cell migration as assessed by the wound-healing assay (with individual 1/2IC for each compound)₅₀And IC₅₀Values were processed for 12, 24, 48, 72 hours). Results are expressed as mean ± SD of at least 3 independent experiments, compared to control (DMSO, 0.5%), + p<0.05；**p<0.01；***p<0.001；****p<0.0001。

Only after 12 hours of incubation, under a microscope, complete cell death was observed for all compounds. At 72 hours, the media was removed from each well before the photographs were taken, as the large number of dead floating cells prevented accurate measurement of cell migration. Compound MC409 shows an early effect on cell migration; after 12 hours, for IC₅₀And 1/2IC₅₀The compound was able to reduce the percentage of cell migration (relative to the 0.5% DMSO control) by about 40%. However, the cells appeared to be degenerated as observed by the slow recovery of cell migrationRecovery in the influence of the compound. After 72 hours of treatment, by using IC₅₀At concentrations, inhibition of cell migration was about 12% less than control. For the other 3 compounds, lower effects were also observed after 24 or 48 hours of treatment. For compound MC408 and MC406 treatments, IC was used₅₀And 1/2IC₅₀Concentration resulted in a similar effect on cell migration, and after 72 hours, cell migration was 20% less than the control. Compound MC421 showed an effect in a concentration-dependent manner. For IC₅₀And 1/2IC₅₀Both concentrations, a slight effect on cell migration over time was noted. A reduction in cell migration was only observed 72 hours after treatment (13% less than control).

In one embodiment, the effect of compounds MC408, MC421, MC412 and MC415 on cell migration was also evaluated in the renal cell carcinoma cell line, Caki-2, at 4 different time points (fig. 2). Cediranib was used as reference drug and their respective ICs were used₅₀Concentration values all compounds were tested. FIG. 2 shows the effect of benzopyranyl compounds on Caki-2 cell line migration as assessed by the wound-healing assay (with respective IC for each compound)₅₀Value treatment for 12, 24, 36, 48 hours). Results were normalized to control (dashed line) and expressed as mean ± SEM. P compared to control (0.5% DMSO)<0.05，**p<0.005，***p<0.002，****p<0.0001. Significance of differences between different groups was determined by student's t-test.

In one embodiment, the effect of preferred benzopyranyl compounds on cell proliferation is evaluated. Using IC' s₅₀And 1/2IC₅₀Values are processed for 786-O cells for 48 hours. The ability of BrdU incorporation during DNA synthesis was measured and the results are shown in figure 3. FIG. 3 shows the effect of benzopyranyl compounds on 786-O cell proliferation after 48 hours of treatment. Results are expressed as mean ± SD of at least 3 independent experiments. P compared to control (0.5% DMSO)<0.05，**p<0.01，***p<0.0005，****p<0.0001. Significance of differences between different groups was determined by student's t-test. Generally, for 786-O cells, benzopyrans induce a significant decrease in cell proliferation in a concentration-dependent manner. To make a rightExceptions were observed for compounds MC409, MC410 and MC 368.

In one embodiment, the compounds show clear effects on cell growth and cell death, in particular the compounds MC406, MC409 and MC 408. This is evident by the morphological changes (cells observed under a microscope) and by the presence of round floating cells.

In one embodiment, to understand the mechanisms leading to cell death, cells were investigated for induction of apoptosis by flow cytometry. MCF-7 and Hs578t cells were incubated with the respective ICs of the compounds₅₀Concentrations were incubated for 12 hours and 24 hours, and 786-O cells were incubated for 24 hours and 48 hours, then double stained with annexin V and Propidium Iodide (PI) to detect phosphatidylserine externalization. Externalization of phosphatidylserine occurs during early apoptotic events. Although annexin V binds to phosphatidylserine, PI only stains cells that have lost their membrane integrity, an experimental procedure that allows differentiation between live, early apoptotic, late apoptotic/necrotic, or necrotic cells. Compounds MC408 and MC421 showed relevant anti-cancer effects by inducing cell death by apoptosis in 3 cell lines at 24h, as shown in figures 9 and 10. In the Hs578t cell line, compounds MC406 and MC409 also induced cell death by apoptosis.

FIG. 4 shows a flow cytometric analysis of MCF-7 cell viability assessed by annexin V/PI assay. FIG. 4A shows the IC of compounds MC408, MC409, MC406 and MC421 treated with 0.5% DMSO (control)₅₀Representative dot plots of MCF-7 cells treated at concentrations for 12 hours and 24 hours. Fig. 4B shows quantification of the percentage of cells in each quadrant of the dot plot. Results were obtained using cells treated with DMSO as control (100%) and expressed as mean ± SEM of 3 independent experiments. Annexin V/PI data were analyzed by two-way ANOVA and Bonferroni post hoc tests. P compared to control (0.5% DMSO)<0.05；**p<0.01；***p<0.001；****p<0.0001. Figure 5 shows a flow cytometric analysis of Hs578t cell viability assessed by the annexin V/PI assay. FIG. 5A shows the use of 0.5% DMSO (control) treatment or IC with Compounds MC408, MC409, MC406 and MC421₅₀Representative dot plots of Hs578t cells treated at concentrations for 12 hours and 24 hours. Fig. 5B shows quantification of the percentage of cells in each quadrant of the dot plot. Results were obtained using cells treated with DMSO as control (100%) and expressed as mean ± SEM of 3 independent experiments. Annexin V/PI data were analyzed by two-way ANOVA and Bonferroni post hoc tests. P compared to control (0.5% DMSO)<0.05；**p<0.01；***p<0.001；****p<0.0001. FIG. 18 shows flow cytometric analysis of 786-O cell viability assessed by annexin V/PI assay. FIG. 18A shows IC treated with 0.5% DMSO (control) or with compounds MC408 and MC421₅₀Representative dot plots of 786-O cells treated at concentrations for 24 hours and 48 hours. Fig. 18B shows quantification of the percentage of cells in each quadrant of the dot plot. Results were obtained using cells treated with 0.5% DMSO as control (100%) and expressed as mean ± SEM of 3 independent experiments. Annexin V/PI data were analyzed by two-way ANOVA and Bonferroni post hoc tests. P compared to control (0.5% DMSO)<0.05；**p<0.01；***p<0.001；****p<0.0001。

In one embodiment, induction of cell death is further investigated by examining the apoptotic pathway by immunoblot analysis of key apoptotic markers. In use of IC₅₀Induced apoptosis was assessed 24 and 48 hours after treatment of cells with compounds using poly (ADP-ribose) polymerase 1(Parp),

caspase

3 and 9 as markers (fig. 6).

Treatment of cells with compounds resulted in cleavage of PARP (fig. 6 and 7), which is the last step of caspase activation and is considered a marker of apoptosis. Cleaved caspase 3 was also observed at two time points. Apoptosis was induced by all compounds as observed by the presence of these markers.

Bim protein was also used and elevated levels were noted for the test compounds but only observed for the Hs578t cell line. BH 3-domain proteins, which are Bim, interact with tubulin and, in an initial phase, the vascular tube is described as sequestering Bim by binding to the dynein light chain, in such a way as to prevent initiation of the apoptotic signal transduction pathway. Upon release from microtubules, Bim migrates to the mitochondria, interacts with some proteins (e.g., Bax, Bcl-2, and Bc-xL), and eventually promotes apoptosis. This suggests that early apoptotic signals are released from cells treated with test compounds, thus affecting the levels of pro-apoptotic and anti-apoptotic proteins involved in mitochondrion-induced apoptosis.

In one embodiment, a cell cycle assay is performed to determine the ability of compounds MC408, MC409, MC406, and MC421 to inhibit cell proliferation. In respective IC₅₀Concentration, MCF-7 and Hs578t cells were treated with compound for 12 hours and 24 hours. In respective IC₅₀786-O cells were treated with compounds MC408 and MC421 for 24 hours and 48 hours. Cell cycle analysis was assessed by DNA content using flow cytometry (fig. 8, 9 and 22). FIG. 8 shows flow cytometry analysis of DNA content of MCF-7 cells. FIG. 8A shows the IC treated with 0.5% DMSO (control) or with MC408, MC409, MC406 and MC421₅₀Representative histograms of cell cycle profiles of MCF-7 cells treated at concentrations for 12 and 24 hours. Figure 8B shows the quantification of cells in different phases of the cell cycle, except for compound MC 409. Results are expressed as mean ± SD of 3 independent experiments. Data were analyzed by two-way analysis of variance and Bonferroni post-hoc testing. P compared to control (0.5% DMSO)<0.05；**p<0.01；***p<0.001；****p<0.0001。

Figure 9 shows flow cytometry analysis of the DNA content of Hs578t cells. FIG. 9A shows the IC treated with 0.5% DMSO (control) or with MC408, MC409, MC406 and MC421₅₀Representative histograms of cell cycle profiles of Hs578t cells treated at concentrations of 12 and 24 hours. Figure 9B shows the quantification of cells in different phases of the cell cycle, except for compound MC 409. Results are expressed as mean ± SD of 3 independent experiments. Data were analyzed by two-way analysis of variance and Bonferroni post-hoc testing. P compared to control (0.5% DMSO)<0.05；**p<0.01；***p<0.001；****p<0.0001。

FIG. 22 showsFlow cytometric analysis of the DNA content of 786-O cells is shown. FIG. 22A shows IC treated with 0.5% DMSO (control) or with MC408 and MC421₅₀Representative histograms of cell cycle profiles of 786-O cells treated at concentrations of 24 and 48 hours. Fig. 22B shows cell quantification in different phases of the cell cycle. Results are expressed as mean ± SD of 3 independent experiments. Data were analyzed by two-way analysis of variance and Bonferroni post-hoc testing. P compared to control (0.5% DMSO)<0.05；**p<0.01；***p<0.001；****p<0.0001。

In one embodiment, the effect of benzopyran based compounds on microtubule dynamics was analyzed. Figure 10 shows the effect of compounds MC408 and MC421 on the microtubule network of Hs578t (A, B) and MCF-7(C, D) cells. Untreated (control), paclitaxel (Hs578t 12 h: 0.25. mu.M, 24 h: 0.01. mu.M; MCF-712/24 h: 1. mu.M) and in IC₅₀Cells treated with compounds MC408 and MC421 for 12(A, C) or 24h (B, D), stained with β -tubulin and counterstained with 4, 6-diamidino-2-phenylindole (DAPI). Microtubules and unassembled tubulin are shown in green. DNA stained with DAPI is shown in blue.

In one embodiment, compound in vivo toxicity assessment is performed using Caenorhabditis elegans (c. A food clearance-based assay was performed in which bacterial consumption over time is representative of insect development, health and fertility (fig. 11). Fig. 11 shows the strategy used in the determination of toxic effects in c. The insects were cultured in liquid medium for 7 days using heat inactivated bacteria (e.coli) OP50) as food source and in the presence of several concentrations of the compounds MC421, MC406, MC369 and MC408, MC409, MC 407. The rate of food consumption between days 3-5 was used as an indicator of insect development, health and fertility, as after day 3, the insect progeny would promote a more rapid reduction in food quantity. Examples are shown for hypothetical compound X. DMSO 1% and 5% were used as negative and positive controls for toxicity, respectively. For this experiment, compounds were dissolved in DMSO 1% (vehicle).

Compounds were tested at several concentrations (up to the highest possible soluble concentration-150 μ M (MC369, 75 μ M)) (fig. 12). Figure 12 shows the in vivo toxicity analysis of MC421, MC406, MC369 and MC408, MC409, MC407 in caenorhabditis elegans (c. The rate of food consumption between days 3-5 was used as an indicator of insect development, health and fertility and compared to 1% DMSO (vehicle, non-toxic). No statistical differences were observed for MC421, MC406, MC369 and MC408, MC409, MC407 (ANOVA followed by Games-Howell post hoc tests). For any compound/compound concentration, no statistical differences in food consumption rates were detected on days 3-5. Even at significantly higher concentrations than those used for in vitro testing, the fact that compounds appear to be well tolerated can be a good predictor of toxicity in mammalian models.

In one embodiment, the safety profile of the compound is evaluated in rodents. FIG. 13 shows the effect of MC408 treatment on C57Bl/6 mice-trial 1. Fig. 13A is a schematic illustration of an experimental timeline. 5-month old female and male animals (n-3/group/sex) were used and injected daily with MC 4083 mg/Kg or vehicle (saline, Tween80 and methyl cellulose) (i.p.) for 7 days. A series of welfare tests (welfare tests) were performed daily. FIG. 13B shows what happens immediately after the 1 st injection; for this analysis, animals were recorded for 10min and the time of inactivity and vertical movement (REARS) were counted by the experimenter. There was no difference between vehicle and MC408 mice. Figure 13B shows the body weight of mice monitored throughout the experiment and there was no difference between the treatment groups. Figure 13D shows the vertical exploration activity of the mice. The activity was measured in a viewing jar (viewing jar) and the number of vertical movements was counted for 5 minutes. Figure 13E shows the horizontal activity of the mice. The activity was recorded in an open field marked with squares and the number of squares was counted while the animals were left to explore freely for 1 minute. There were no differences in the exploration activities. Figure 13F shows indirect measurements of anxiety in mice. To indirectly measure anxiety, the number of animal stool pellets produced during the performance program was counted and no difference was observed between groups. In fig. 13G and H, water and food intake were analyzed, respectively. Each day, 0.300g of food and 200mL of water were placed in each cage. At the end of the test (day 7), the food was weighed and the water was measured, and there was no difference in the two parameters. Data are expressed as mean ± SE.

FIG. 14 shows the effect of MC408 treatment on C57Bl/6 mice. A series of welfare tests were performed to evaluate signs of toxicity of treatment with MC408 when compared to vehicle animals. All parameters evaluated were scored as normal or abnormal, present or absent, and equally for all animals (MC408 and vehicle), indicating that the compound had no effect on mouse welfare.

In one embodiment, the effect of treatment on liver function in C57Bl/6 mice was analyzed. FIG. 15 shows the effect of MC408 treatment on enzymatic liver function in C57Bl/6 mice. Aspartate aminotransferase (AST/TGO) and alanine aminotransferase (ALT/TGP) were measured in the serum of females (fig. 15A) and males (fig. 15B) using standard techniques. Blood was collected at the end of the experiment (day 7). Within each sex, no statistical differences were found between compound-and vehicle-treated animals. Notably, one male animal in the MC408 treated group showed higher levels of both AST and ALT compared to the other two animals in the same group. Data are expressed as mean ± SE.

In one embodiment, the effect of compound treatment on C57Bl/6 mice was analyzed-trial 2. FIG. 16 shows the effect of MC408 treatment on C57Bl/6 mice-trial 2. Fig. 16A is a schematic illustration of an experimental timeline. 3-month old female and male animals (n ═ 6/group/sex) were used and injected daily for 7 days with MC 4083 mg/Kg or vehicle (saline, Tween80 and methyl cellulose) (i.p.). In this experiment, the number of animals per group was increased and the experiment was conceived to collect blood before and after treatment. Fig. 16B shows the body weight of the mice. Body weight was monitored throughout the experiment and there was no difference between groups within each gender. Data are expressed as mean ± SE.

In one embodiment, the CAM model is used to analyze in vivo therapeutic efficacy studies of compounds. Figure 17 shows the in vivo therapeutic effect of MC408 and MC421 on breast cancer Hs578t cell line. FIG. 17A shows in ovo and out ovo CAM assaysRepresentative photographs (× 10 magnification) (on days 13 and 17). Figure 14B shows the percentage of tumor growth. Results are expressed as mean percentage of tumor growth ± SD from day 13 to day 17 of development<0.0001. Data were analyzed by one-way anova test. Control (DMSO 0.5%), MC408(2 × IC)₅₀0.094 μ M) or MC421(2 × IC)₅₀0.070 μ M) eggs were treated.

In one embodiment, induction of cell death is further investigated by examining the apoptotic pathway by immunoblot analysis of key apoptotic markers. In use of IC₅₀Induced apoptosis was assessed 24 and 48 hours after 786-O cells were treated with compounds using poly (ADP-ribose) polymerase 1(Parp), heat shock protein 90(Hsp90), c-Jun N-terminal kinase (JNK), p53, BH3 interaction-domain death agonist (Bid), and p21 as markers (FIG. 19).

In one embodiment, the expression of rapamycin (mTOR), extracellular-signaling-regulated kinase (ERK), vascular endothelial growth factor receptor 2 (VEGFR) is assessed²) The mammalian targets of Endothelial Growth Factor Receptor (EGFR), protein kinase B (Akt), phosphatase and tensin homolog (PTEN), 5' adenosine monophosphate-activated protein kinase (AMPK), pyruvate dehydrogenase kinase 1(PDK1), nuclear factor kB (NF-kB), p18, c-Myc, Hsp90 and the 40kDa proline-rich Akt substrate (PRAS40) protein, further investigated the interaction with RTK receptors and mTOR/PI3K/Akt pathway. These markers were evaluated 6 hours after treatment with cediranib, an effective inhibitor of vascular endothelial growth factor used in the treatment of renal cell carcinoma, and with compounds MC350, MC412, MC415, MC408 and MC421 using a unique dose of 2 μ M (fig. 20).

Treatment of 786-O cells with several compounds resulted in decreased levels of phosphorylated-mTOR and AKT protein. Furthermore, in some cases, EGFR and VEGFR when compared to controls and cediranib²Both showed reduced levels. Some of the test compounds affect the mTOR/PI3K/AKT pathway and thus interfere with different protein markers.

VEGF and EGFR are tyrosine kinase (RTK) receptors specific for the Vascular Endothelial (VEGF) and Endothelial (EGF) growth factor families, which play important roles in tumor growth and metastasis. Through a variety of other proteins associated with phosphorylated tyrosines, their autophosphorylation stimulates downstream activation and signaling. These downstream signaling proteins initiate several signaling cascades, including the Akt pathway, leading to gene expression, cell proliferation and migration, angiogenesis and vasculogenesis. In addition, mTOR regulates cell growth, proliferation, and metabolism. Its activity is controlled by two polyprotein complexes mTORC1 (sensitive to rapamycin) and mTORC2, which are considered to be either tolerated or inhibited only when provided at high doses for a sustained period of time. Second generation dual mTOR inhibitors have been developed and the most important advantages of those new drugs are a significant reduction of mTORC2 blocked AKT phosphorylation and a better inhibition of mTORC 1. Considering the results, a decrease in RTK, mTOR and AKT expression indicates a decrease in key regulators for all functions described above.

In one embodiment, the CAM model is used to analyze the in vivo therapeutic efficacy of a compound. FIG. 21 shows the in vivo therapeutic effect of MC408 and MC421 on renal cancer 786-O cell line. Figure 21A shows representative photographs of in ovo and out ovo CAM assays (x 10 magnification) (on days 13 and 17). Figure 21B shows the percentage of tumor growth. Results are expressed as mean percentage of tumor growth + -SD from day 13 to day 17 of development, p compared to control<0.0001 (statistical analysis using one-way anova test). Control (DMSO 0.5%), MC408(2 × IC)₅₀0.128 μ M) or MC421(2 × IC)₅₀0.154 μ M) treated eggs.

In one embodiment, the effect of cediranib, rapamycin, and benzopyranyl compounds on cell proliferation was evaluated. Using IC' s₅₀And 1/2IC₅₀Values A498 (parental and rapamycin-tolerant) and Caki-2 (parental and cediranib-tolerant) cells were treated for 24 and 48 hours. The ability of BrdU incorporation during DNA synthesis was measured and the results are shown in fig. 23 and 24. FIG. 23 shows the effect of benzopyranyl compounds on A498 (parental and rapamycin-tolerant) cell proliferation after 24 and 48 hours of treatment. FIG. 24 shows that benzopyranyl compounds were resistant to Caki-2 (parental and cediranib) cells after 24 and 48 hours of treatmentThe effect of proliferation. Results are expressed as mean ± SD of at least 3 independent experiments.

Generally, benzopyrans induced a significant decrease in cell proliferation in a concentration and time dependent manner for parental or resistant a498 cells, in particular the compounds MC408, MC421 and MC413 (fig. 23).

For parental and drug-resistant Caki-2 cells (FIG. 24), benzopyrans MC421, MC350, and MC413 were able to reduce cell proliferation in a concentration and time dependent manner.

In one embodiment, the interactions associated with metabolic features were further investigated by evaluating total extracellular-signal-regulated kinase (ERK) and its phosphorylated form, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase 2(HK2), lactate dehydrogenase a (ldha), hypoxia-inducible factor 2 α (Hif2 α), monocarboxylate transporter 1(MCT1), pyruvate kinase isozyme (PKM), 6-Phosphofructokinase (PFKL), heat shock protein 90(Hsp90), and c-Myc protein. IC in rapamycin and cediranib, Compounds MC350, MC413, MC408 and MC421₅₀These markers were evaluated after 48 hours of treatment of A498 parental (P) and tolerance (R) cells. Protein levels in lysates were detected by immunoblotting using a 10% polyacrylamide gel (fig. 25).

Treatment of a498 cells with different compounds resulted in a reduction in some protein levels. Indeed, rapamycin-tolerant cells showed significant reductions in GAPDH, Hif2 α, c-Myc, and Hsp90 protein expression levels compared to controls and even to parental cells. In view of the results of the present invention, benzopyrans alter the metabolic response of cancer cells, even when resistant to currently available RCC therapies.

In one embodiment, the ability of a compound to inhibit angiogenesis in vivo is analyzed using a CAM model. To evaluate the effect on angiogenesis, sterilized 5-mm diameter filter disks were immersed at a fixed concentration (2 × IC)₅₀Values) of benzopyrans MC408 and MC421 or 0.5% DMSO (control) were placed in culture media on the vascular zone of the CAM. Figure 26 shows representative photographs of in ovo and out ovo CAM assays (x 10 magnification) (on days 13 and 17).

After 4 days of treatment with the novel benzopyrans, the formation of new blood vessels decreased with benzopyrans MC408 and MC 421. In addition, rupture of pre-existing blood vessels was observed (black arrows in fig. 26), indicating that the compound inhibited angiogenesis in the CAM model.

In one embodiment, the in vivo therapeutic efficacy of compound MC408 is analyzed using a mouse model. Figure 27 shows the in vivo therapeutic effect of MC408 in orthotopic breast cancer mouse xenograft model using TNBC cell line MDA-MB-231. MDA-MB-231 cells were injected into the mammary fat pad of NSG mice (n ═ 6 or 7 per group). Treatment was administered 3 days after implantation for one week. NT is vehicle; the dosage is 1-3 mg/kg; the dose 2 is 10 mg/kg; the dose is 3-50 mg/kg. P < 0.05; p < 0.01; p <0.0001 (compared to NT group; two-way analysis of variance post Tukey test or unpaired t test with Welch correction).

In an orthotopic breast cancer xenograft mouse model, benzopyran MC408 significantly reduced tumor volume (fig. 27A) and weight (fig. 27B) in a dose-dependent manner. At a dose of 50mg/kg, both tumor volume and weight were reduced by greater than 40% compared to the vehicle group. Importantly, the animals showed no weight loss and also no other side effects for the tested treatment regimens during the entire experiment.

In one embodiment, the 3 human breast cancer cell lines Hs578T, MDA-MB-468 and MCF-7 and the normal breast cell line MCF-10A are obtained from ATCC (American type culture Collection). The cancer cell lines were cultured in Darber's modified eagle's medium, 4.5g/l glucose (DMEM, Gibco), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% antibiotic solution (penicillin-streptomycin, Gibco). Culturing a normal cell line in a dartbuck modified eagle medium: nutritional mix (Nutrient mix) F-12(DMEM/F12, Gibco) supplemented with 5% heat-inactivated fbs (Gibco), 1% antibiotic solution (penicillin-streptomycin, Gibco), 1% sterol hormone (hydrocortisone, Sigma-Aldrich), 0.1% peptide hormone (insulin, Sigma-Aldrich) and 0.01% protein complex (Cholera Toxin, Gibco). Two different human acute myelocytic leukosis were also usedLeukemia (AML) cell lines HL-60(FAB M2) and KG-1 (erythroleukemia-FAB M6) and a human lymphocytic leukemia (ALL) cell line Jurkat (T cell type). These 3 cell lines were obtained from the German Collection of microorganisms and cell cultures (

German is Deutsche Sammlung von Mikroorganismen und Zellkulturen). Cells were supplemented with 10% heat-inactivated FBS ((FBS))

Merck Millipore) and 1% antibiotic/antimitotic mixture (

) RPMI1640 medium (A), (B)

Merck Millipore). Also used were 3 human Glioblastoma (GBM) cell line models, two established and commercially available cell lines (U87MG and GAMG, friendly gift from Rui m. reis) and one primary GBM culture (GL 18). GBM cells were cultured in DMEM (Biochrom-Merck Millipore), all supplemented with 10% FBS. The renal cell carcinoma cell lines A498, 786-O, Caki-2 and HK2 were obtained from ATCC. Cancer cell line A498 in MEM Medium (

Merck Millipore) 786-O was cultured in RPMI1640 medium (

Merck Millipore) and Caki-2 in Mc Coys medium (C)

Merck Millipore) and HK2 in RPMI1640 medium (

Merck Millipore). All mediaSupplemented with 10% heat-inactivated FBS (

Merck Millipore) and 1% antibiotic/antimitotic mixture (

). All cells were incubated at 37 ℃ and 5% CO₂Growth was performed in a humidified incubator. For all assays, DMSO (dimethyl sulfoxide, Sigma-Aldrich) control was used.

In one embodiment, a cell viability assay is performed. MCF-7, Hs578t and MCF-10A cells were plated in 96-well plates in triplicate at 3000 cells per mL or 5000 cells per mL (100. mu.L/well) for MDA-MB-468. Cells were then allowed to adhere in complete medium for a period of 18-20 hours. Subsequently, cells were treated with 7 different concentrations (0.1 to 40 μ M or 5 to 60 μ M) of compound or control in fresh medium. HL-60, KG-1 and Jurkat cell lines were plated at 50.000 cells per 100 μ L per well in 96-well culture plates and treated with different concentrations (0.001 to 2 μ M) of the compounds described (or controls). Cells were plated in 96-well plates at an initial density of 2000 cells/well (for GAMG), 4000 cells/well (for GL18) and 6000 cells/well (for U87MG), repeated three times, and allowed to adhere for 18-20 hours. Cells were treated with different concentrations of compound (0.005 to 100 μ M) or with controls. In 96-multiwell culture plates (100. mu.L/well), A498 and 786-O cells were plated at 2000 cells per mL, Caki-2 cells at 3000 cells per mL and HK2 cells at 2000 cells per mL, repeated three times, and allowed to adhere in complete medium over a period of 18-20 hours. Cells were then treated with 7 different concentrations (0.1 to 40 μ M or 5 to 60 μ M) of compound or control in fresh medium. After 72h incubation, an mts (promega) reduction assay was performed according to the manufacturer's instructions to indirectly assess the percentage of viable cells by metabolic cell viability. At 37 ℃ and 5% CO₂Absorbance at 490nm was measured after incubation with MTS in a humidified atmosphere for 1-2 hours. For RCC cell linesSulforhodamine B assay was used according to the manufacturer's instructions. Data were log-transformed and the concentration of each compound that reduced viable cell number to 50% relative to control (IC) was calculated using GraphPad Prism 6 software₅₀)。

In one embodiment, the IC of all compounds for MCF-7 and MCF10A cell lines was used using the following mathematical formula₅₀Value, calculating the Selectivity Index (SI) value:

SI＝(IC₅₀normal cell line-IC₅₀Cancer cell line)/IC₅₀Cancer cell lines

For SI values >1, cytotoxicity was higher for cancer cell lines than for non-neoplastic cell lines.

In one embodiment, cell migration is assessed by a wound-healing assay that mimics the process of cell migration during wound-healing in vivo. The method is based on the following processes: scratch marks were prepared, wound in a monolayer of simulated cells, images were recorded at the beginning and at regular intervals during the process of cell migration to close the wound and the images were compared to quantify the rate of cell migration. MCF-7 and Caki-2 cells were cultured at 9.0X 10, respectively⁵And 3.0X 10⁵Plates were plated per 2mL density in 6-well plates and at 37 ℃ in 5% CO₂Growth was carried out overnight in a humidified atmosphere. A 200 μ L pipette tip was used to make two passes of scratch in the confluent cell layer. Cells were gently washed once with 500 μ L PBS. With respective IC₅₀、1/2IC₅₀MCF-7 cells were treated with either compound or 0.5% DMSO (control) for 72 hours. Specific wound sites (4 sites per wound) were photographed at 0,12, 24, 48 and 72 hours. For Caki-2 cells, the individual IC of each compound was used₅₀Treatments including cediranib or 0.5% DMSO (control) were performed for 48h and photographs were taken at 0,12, 24, 36 and 48 hours. Images were acquired at 100 x magnification using an Olympus IX51 inverted microscope equipped with an Olympus DP20 digital camera system. Evaluation of migration distance was performed 5 times using BeWound 1.7.1 and percentage of cell migration normalized to control was plotted using GraphPad Prism 6 software. 3 independent experiments were performed for each compound. Determination of different groups by student's t-testThe difference between them is significant.

In one embodiment, a proliferation assay is performed. 786-O cells were plated at 7000 cells/10. mu.L in 96-well plates and allowed to stand at 37 ℃ in 5% CO₂Incubate overnight in a humidified atmosphere. Thereafter, with IC₅₀Or 1/2IC₅₀Adherent cells were treated with compounds MC350, MC408, MC412, MC415, MC409, MC410, MC413 and MC369 at concentrations or with 0.5% DMSO (control) for 48 h. After incubation, cells were labeled by adding 10. mu.l/well of BrdU labeling solution (final concentration: 40. mu.MBrdU). The cells were then re-incubated for 16h to allow BrdU to be introduced into the proliferating cell DNA, replacing thymidine. After labeling, the medium was removed, the cells were fixed and the DNA denatured by incubation with 200 μ l of FixDenat solution at room temperature. Denaturation of DNA is necessary for the antibody conjugate to bind to the incorporated BrdU. After removal of FixDenat, and 100. mu.l of anti-BrdU-POD antibody was incubated at room temperature for 90 min. The anti-BrdU-POD antibody is bound to the incorporated BrdU in newly synthesized cellular DNA. The antibody conjugate was removed and the wells were washed three times with washing solution. The immune complexes were detected by addition of substrate solution (100. mu.l/well) and the plates were incubated at room temperature until the development was satisfactory for photometric detection (5-10 min). By adding 25. mu.l of 1M H to each well₂SO₄And gently mixed to terminate the substrate reaction. The reaction product was quantified by measuring the absorbance at 450nm (reference wavelength: 690nm) in a microplate reader (Tecan Infinite M200). All the above steps were performed without cells, using a blank test at each experimental time point. The results of at least 3 independent experiments (four replicates) were evaluated using GraphPad Prism 5 software.

In one embodiment, protein extraction and immunoblot analysis are performed. MCF-7 and Hs578T cells were grown in T25 flasks and 786-O cells were grown in 6-well plates at respective IC's when the cells reached 70-80% confluence₅₀Values were treated with compound or cells were treated with 0.5% DMSO (control). Cells were treated for 24 hours and 48 hours. Specifically, for RTK and mTOR pathway analysis, 786-O cells were starved for 2 hours and then treated with a unique dose of 2 μ M cediranib or compound orTreated with 0.5% DMSO (control). After treatment, adherent and floating cells were collected by scraping and centrifuged at 2000rpm at 4 ℃ for 10 min. The particles were resuspended in PBS and centrifuged again (1200 rpm; 5min, 4 ℃). The supernatant was discarded and the pellet was placed in lysis buffer (50mM Tris pH 7.6-8, 150mM NaCl, 5mM EDTA, 1mM Na)₃VO₄10mM NaF, 1% NP-40, 1% Triton-X100, and 1/7 protease inhibitor cocktail (Roche Applied Sciences)) and incubated on ice for 20 minutes. The lysate was centrifuged at 14000rpm for 15 minutes at 4 ℃; then, the supernatant was collected for protein concentration determination using DC protein assay kit (BioRad).

Briefly, 30 μ g of total protein from each sample was separated in 10%, 12% or 15% polyacrylamide gels (100V) and then transferred to nitrocellulose membranes (100V, 30 min). The membrane was blocked with a solution of 5% milk in 1 × TBS for 60 minutes and then incubated with a specific first antibody at 4 ℃ overnight (rabbit anti-PARP antibody (Cell Signaling, #9542), 1: 10005% milk; mouse anti-caspase-9 antibody (Cell Signaling, #9508), 1: 5005% milk; rabbit anti-caspase-3 antibody (Cell Signaling, #9665), 1: 5005% milk; mouse anti-Bax antibody (Santa Cruz Biotechnology, sc-7480), 1: 5005% milk; mouse anti-Bax antibody (Santa Cruz Biotechnology, sc-8392), 1: 5005% milk; rabbit anti-Bim antibody (Cell naling, #2933), 1: 10005% BSA; rabbit anti-Bid antibody (Cell Signaling, # 2002% rabbit anti-Bim antibody; 1: 855% BSA; rabbit anti-Betacam antibody; 10008584; Calcamb-3505; Ab-2784; Ab-Ab), cat #386041), 1: 5005% BSA; rabbit anti-c-Myc antibody (Cell Signaling, #5605), 1: 10005% BSA; rabbit anti-p 53 antibody (Cell Signaling, #2527), 1: 10005% BSA; rabbit anti-phospho-p 53 antibody (Cell Signaling, #2521), 1: 5005% BSA; rabbit anti-p 21 antibody (Cell Signaling, #2947), 1: 10005% BSA; rabbit anti-JNK antibody (Cell Signaling, #92525), 1: 5005% BSA; rabbit anti-phospho-JNK antibody (Cell Signaling, #46685), 1: 5005% BSA; rabbit anti-mTOR antibody (Cell Signaling, #2983), 1: 10005% BSA; rabbit anti-phospho-mTOR antibody (Cell Signaling, #5536), 1: 5005% BSA; rabbit anti-PRAS 40 antibody (Cell Signaling, #2691), 1:10005% BSA; rabbit anti-VEGFR²Antibody (Cell Signaling, #2479), 1: 5005% BSA; rabbit anti-phospho-VEGFR²(Tyr1175) antibody (Cell Signaling, #2478), 1: 5005% BSA; rabbit anti-EGFR antibody (Cell Signaling, #4267), 1: 15005% BSA; rabbit anti-phospho-EGFR (Tyr1068) antibody (Cell Signaling, #2234), 1: 15005% BSA; mouse anti-phospho-NF-kB p65(Ser536) antibody (Cell Signaling, #3036), 1: 10005% BSA; rabbit anti-AMPK α antibody (Cell Signaling, #2532), 1: 10005% BSA; rabbit anti-phospho-AMPK α antibody (Thr172) (Cell Signaling, #2535), 1: 10005% BSA; rabbit anti-AKT antibody (Cell Signaling, #4691), 1: 10005% BSA; rabbit anti-phospho-AKT (Thr308) antibody (Cell Signaling, #13038), 1: 10005% BSA; rabbit anti-PTEN antibody (Cell Signaling, #9559), 1: 10005% BSA; rabbit anti-phospho-PTEN (Ser380) antibody (Cell Signaling, #9551), 1: 10005% BSA; rabbit anti-phospho-PDK 1(Ser241) antibody (Cell Signaling, #3438), 1: 10005% BSA; rabbit anti-phospho-p 38(Thr180/Tyr182) antibody (Cell Signaling, #4511), 1: 10005% BSA; rabbit anti-p 44/42MAPK (Erk1/2) antibody (Cell Signaling, #4695), 1: 10005% BSA; rabbit anti-phospho-p 44/42MAPK (Thr202/Tyr204) (phospho-Erk 1/2) antibody (Cell Signaling, #4370), 1: 10005% BSA; rabbit anti- β -tubulin antibody (Abcam, ab6046), 1: 100005% BSA; mouse anti-GAPDH antibody (Santa Cruz Biotechnology, sc-32233), 1: 10005% BSA; mouse anti-HK 2(Abcam, ab104836), 1: 10005% BSA; mouse anti-LDHA antibody (Santa Cruz Biotechnology, sc-137243), 1: 10005% BSA; rabbit anti-Hif 2 α antibody (Cell Signaling, #36169), 1: 10005% BSA; rabbit anti-MCT 1 antibody (Santa Cruz Biotechnology, sc-365501), 1: 10005% BSA; rabbit anti-PKM (Abcam, ab38237), 1: 10005% BSA; rabbit anti-PFKL (Abcam, ab37583), 1: 10005% BSA and mouse anti- β -actin antibody (Santa Cruz Biotechnology, # E1314), 1: 5005% milk). After washing with 0.1% tween 20 for 5min (twice) and another 15min (once), the blots were incubated with the respective secondary antibodies for 1h at room temperature (Apoptosis Antibody sampling Kit (Apoptosis Antibody Sampler Kit) -Cell Signaling (# 9915): goat-anti-rabbit IgG-HRP (7074) and horse-anti-mouse IgG-HRP (7076) secondary antibodies, 1: 20005% milk, Cell Signaling; and rabbit-anti-rat IgG-HRP secondary antibody (Abcam, ab6734), 1:30000, 5% BSA). After washing with TBS/0.1% Tween 20 for 5 minutes (twice) and another 15 minutes (once), by chemiluminescence WesternBright^TMThe Sirius kit (Advansta) detected immunoreactive bands on a ChemiDoc XRS + system (BioRad). Immunoblot quantification was performed by Quantity One 4.6.9.

In one embodiment, cell cycle distribution by flow cytometry is performed. MCF-7, Hs578t and 786-O cells were seeded into 6-well culture plates. Cells were allowed to adhere in complete DMEM (for MCF-7 and Hs578t cells) and complete RPMI (for 786-O cells) medium for a period of 18-20 hours and IC was used₅₀Test compounds at concentrations or 0.5% DMSO (control) were treated for 12 hours and 24 hours. Each experiment was repeated three times. Floating and adherent cells were collected and pooled by centrifugation and fixed with cold ethanol (70%). Resuspend cells in PBS; after centrifugation and removal of the supernatant, it was resuspended in a solution containing PBS, PI (50. mu.g/mL, P1304MP, Invitrogen), RNase A (20mg/mL,12091-021, Invitrogen) and Triton X100. After a final incubation for 1 hour at 50 ℃ protected from light, FACS LSRII flow cytometer (BD) was used

) PI signals were measured with a 488nm excitation laser, recorded and FACS Diva was used as the acquisition software. Using FlowJo 7.6 (Tree)

) The software analyzed the percentage of cells in each phase. 3 independent biological replicates were performed.

In one embodiment, MCF-7, Hs578t, and 786-O cells are seeded in 6-well culture plates and allowed to adhere in complete DMEM (for MCF-7 and Hs578t cells) and complete RPMI (for 786-O cells) media within 18-20 hours to detect apoptosis by flow cytometry analysis. Using IC' s₅₀Cells were treated with test compound at concentrations or 0.5% DMSO (control) for 12 hours and 24 hours. Each experiment was repeated three times. Floating and adherent cells were collected and pooled by centrifugation. Removing the supernatantThen 1mL of binding buffer was added. mu.L of FITC annexin V (556419, BD Pharmingen) and 30. mu.L of PI (50. mu.g/mL, P1304MP, Invitrogen) were added to the cell pellet. The samples were incubated at room temperature for 15min in the dark. An additional 200 μ L of binding buffer was added to each sample. Using FACS LSRII flow cytometer (BD)

) The PI signal was measured with a 488nm excitation laser. Annexin V signals were collected by 488nm blocking filter, 550nm long-pass dichroic mirror (long-pass dichroic) with 525nm band-pass. Signals were recorded and FACS Diva was used as the acquisition software. Using FlowJo 7.6 (Tree)

In one embodiment, each compound was tested for toxicity based on a food clearance assay using caenorhabditis elegans (c.elegans) using Bristol strain N2 (supplied by CGC, funded by the NIH Office of Research Infrastructure project-P40 OD 010440). The assay was performed in liquid culture on 96-well plates (Voisine et al 2007; Teixeira-Castro et al 2015). Each well included the following (final volume 60 μ Ι _): about 20 insects in egg phase, OP50 bacteria to a final OD of 0.6-0.8(595nm) and each compound at the appropriate concentration. The insects were grown at 180rpm/20 ℃ for 7 days under continuous shaking (Shell lab Si series incubator) and the absorbance (OD595) was measured daily on a microplate reader (Tecan Infinite M200). The effect of compounds on caenorhabditis elegans (c. elegans) physiology was monitored by the e.coli (e. coli) food suspension consumption rate (fig. 14). Age-synchronized worm eggs were obtained by "egg preparation (egg prep)" as follows: adults were treated with alkaline hypochlorite solution (20% bleach, 25% 1M NaOH) for 6 minutes, then centrifuged and washed 2 times in M9 buffer. The egg granules were then resuspended in S-medium to obtain the appropriate egg concentration and then transferred to 96-well plates. OP50 bacteria were prepared by inactivating the culture overnight (37 ℃, 180rpm, Luria broth) by 4 freeze-thaw cycles. OP50 was resuspended in supplemented S-medium (cholesterol, streptomycin, penicillin and nystatin) prior to use. Compounds were prepared in 100% DMSO (sigma) and diluted up to the test concentration of 1% DMSO to prevent solvent toxicity. For each compound, several concentrations (150-10 μ M) were tested, except for MC369(75-10 μ M) due to solubility issues.

For each compound, several concentrations were used and the corresponding OD595 consumption rates (slopes) were calculated on days 3-5. In two independent experiments, compounds were tested in worms grown in 96-well plates (plateau layout. xls, Supporting Information). For statistical analysis, data from two experiments were mixed together and ANOVA (Brown-Forsythe robust test for homogeneity of mean) was applied using IBM SPSS, followed by a Games-Howell post hoc test to compare different concentrations and 5% DMSO (wt/v) for each compound to a control (1% DMSO (wt/v)).

In one embodiment, C57/Bl6 female and male mice (n ═ 3/group/sex) were injected intraperitoneally daily with MC408(3mg/Kg) or vehicle (saline, Tween80 and methylcellulose) for 7 days to determine toxicity of the compounds in the mouse model. A series of tests were performed daily to assess the welfare of the mice. Immediately after the first drug administration, the animals were videoed and the immediate effect of MC408 was recorded. The number of vertical movements and the time of inactivity were recorded. Water and food intake during the 7 day experiment was evaluated by adding fixed amounts of food and water to the animal's rearing cage on the first day and measuring the remaining amount on day 7. Body weight was assessed daily in a dynamic mouse scale. Analyzing the horizontal and vertical activity to evaluate the animal's intrinsic exploratory behavior; these were measured in the open field of the marked squares for 1 minute and in the observation tank for 5 minutes, respectively. In addition, as a measure of anxiety, the number of fecal pellets was also counted while the animals were subjected to a daily behavioral assessment protocol. The experimenter applied a series of tests more relevant to animal welfare, including the following measurements: (i) body posture and bending; (ii) spontaneous activity; (iii) a respiration rate; (iv) eyelid opening and reflectance; (v) grab the skin to analyze dehydration; (vi) dressing up; (vii) standing the fur upright; (viii) shaking; (ix) limb clasping and (x) response to metastasis. On day 7, animals were euthanized. All animals were deeply anesthetized (ketamine hydrochloride (150mg/kg) and medetomidine (0.3mg/kg)) and perfused with a saline solution (nacl0.9% (wt/v)) heart. Blood was collected from the vena cava, centrifuged at 13.000rpm for 10 minutes and the plasma transferred to a new tube and stored at-80 ℃ until further processing for enzymatic liver function measurements using standard techniques. Organs (kidney, intestine, stomach, brain, liver, ovary/testis) were harvested and placed in tubes containing 4% paraformaldehyde and further processed for paraffin embedding and pathology analysis.

In one embodiment, a CAM assay is performed. Chicken zygotes were grown at 37 ℃ and the eggshells were windowed after puncturing the air chamber on day 3 of development. The window was sealed with BTK tape and returned to the incubator. Hs578t cells (2X 10) at day 9 of development⁶One cell) or 786-O cell (3.5X 10)⁶Individual cells) and Matrigel (10 μ L) were placed on the CAM, allowing tumors to form and the eggs to be tapped and returned to the incubator. On day 14 of development, tumors were photographed. Treatment groups received 20 μ L of 2 × IC₅₀MC408 or MC421 in complete DMEM (for Hs578t cells) or complete RPMI (for 786-O cells) medium at concentrations and controls received 20 μ Ι _ of 0.5% DMSO in complete DMEM or RPMI; these were added over the formed tumor. After 72 hours of treatment (day 17 of development), the tumors were again photographed in ovo. Chicken embryos were sacrificed for 10 minutes at-80 ℃ and individual CAMs were fixed with 4% paraformaldehyde before being photographed again in vitro.

In one embodiment, 0.6X 10 is used⁶A mixture of MDA-MB-231 breast cancer cells and matrigel (ratio 1:1) was injected into NSG (NOD Scid Gamma) female mice (3-6 months) in the mammary fat pad. Mice were randomly assigned to treatment groups (at least 6 mice/group) starting 3 days after implantation with 3 independent doses of compound MC408 (dose 1 ═ 3mg/kg, dose 2 ═ 10mg/kg and dose 3 ═ 50mg/kg) or vehicle (PBS 1 × and Matrigel, ratio 1:1) and administered daily for a total of 7 days. Mice were maintained under standard laboratory conditions. Measuring animal tumor size(calculated by measuring the two largest sides and applying the formula: v ═ 3.14 xl 1 xl 1 xl 2/6; L1 is the largest side and L2 is the other) and recorded periodically. On day 47 post-implantation, animals were sacrificed and tumors excised. All animal procedures were performed according to the laboratory guidelines for animal protection and use (European directive 2010/63/EU).

In one embodiment, the reaction of all compounds was monitored by Thin Layer Chromatography (TLC) using silica gel60 plates (Macherey-Nagel) at 0.2mm and a fluorescent indicator. UV chambers with 254nm lamps (CN-6Vilber Lourmat) were used for their display. Silica gel (particle size) from MN Kieselgel60(230ASTM) was used<0.063mm) was subjected to dry flash chromatography. For the reactions using temperatures, suitable magnetic stirring was used and electric furnace stirrer IKAMAG RCT was used at different temperatures according to the specific procedure. The solvent was evaporated in a Buchi RE 11 rotary evaporator with vacuum and different bath temperatures. At 25 ℃ and using deuterated dimethyl sulfoxide (DMSO-d6) as solvent, at Bruker Avance III (for¹H NMR at 400MHz and for¹³C NMR at 100 MHz). Chemical shifts were recorded in parts per million (ppm) using the residual solvent peak as an internal standard. IR spectra were recorded in FT-IR Bomem MB 104 using a paraffin paste and NaCl cell (NaCl cell). The melting point was determined in a Stuart SMP3 apparatus and no correction was made.

Elemental analysis was performed on a LECO CHNS-932 instrument.

2-imino-8-methoxy-2H-benzopyran-3-amine (4) and benzopyran derivatives M1159, M955, M1220, M1221 and M1143 can be synthesized by the above-mentioned method.

In one embodiment, the synthesis of 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) is performed. Concentrated HCl (1.1 equiv.) was added to 2-imino-8-methoxy-2H-chromen-3-amine (4) (0.150 mg; 0.79mmol) in 1mL CH₃Orange solution in CN. Direct precipitation was observed and the reaction mixture was stirred at room temperature10-15 minutes. An orange solid was isolated by simple filtration and identified as 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5). An orange solid; the yield is 91 percent; mp is more than 300 ℃;¹H NMR(400MHz,DMSO-d₆)

3.93(s,3H),7.12-7.15(m,2H),7.18(dd,J＝8.0,1.2Hz,1H),7.32(t,J＝8.0Hz,1H),11.48(s,1H)；¹³C NMR(75MHz,DMSO-d₆)56.02,110.13,113.52,117.42,122.21,126.39,130.57,135.33,146.55,161.15, respectively; IR (Paraffin paste)

3322,3192,1678,1654,1600,1576,1552,1460cm^-1；C₁₀H₁₁N₂O₂Cl.0.2H₂Theoretical value of O: c, 52.16; h, 4.96; and N, 12.17. Experimental values: c, 52.06; h, 4.77; and N, 12.40.

In one embodiment, benzopyrano [2,3-d ] is synthesized]Imidazole. Aldehyde (1) (1.1-1.7 equiv.) was added to 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) (0.25-0.35mmol) in CH₃CN (1-2mL) and the suspension was stirred at 60 ℃ for 24-48 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃The solid was washed with an aqueous solution of (0.05M), filtered and washed with water, resulting in the production of a pure product.

In one embodiment, 2- (4-fluorophenyl) -5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazole (MC 409). 4-fluorobenzaldehyde (1) (0.0375 mg; 0.30mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-imido chloride (5) (0.047 mg; 0.21mmol) in CH₃CN (1.6mL) and the suspension was stirred at 60 ℃ for 32 hours. Filtering the solid with CH₃CN was washed and identified as pure product. A beige solid; the yield is 98 percent; mp191-192 ℃;¹H NMR(400MHz,DMSO-d₆)3.82(s,3H),4.12(s,2H),6.94(dd,J＝8.4,1.8Hz,1H),6.86(dd,J＝7.4,1.8Hz,1H),7.00(t,J＝7.8Hz,1H),7.25-7.32(m,2H),7.86-7.92(m,2H),12.44(s,1H)；¹³C NMR(75MHz,DMSO-d₆)23.64,55.75,103.64,110.48,115.77(J＝21.7Hz),119.31,121.82,122.59,126.29(J＝8.3Hz),127.19(J＝3.2hz),138.88,141.25,148.19,148.38,161.78 (J243.4 Hz); IR (Paraffin paste) 3348,1700,1650,1608,1578,1538,1500,1461cm^-1；C₁₇H₁₃N₂O₂Theoretical value of F: c, 68.91; h, 4.42; and N, 9.45. Experimental values: c, 68.89; h, 4.65; n, 9.56.

In one embodiment, 2- (4-bromophenyl) -5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazole (MC 408). 4-Bromobenzaldehyde (0.0737 mg; 0.40mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) (0.0758 mg; 0.34mmol) in CH₃CN (2mL) and the suspension was stirred at 60 ℃ for 24 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃The solid was washed with an aqueous solution (0.05M; 2mL), filtered and washed with water, resulting in the production of pure product. A yellow solid; the yield is 99 percent; mp215-217 ℃;¹HNMR(400MHz,DMSO-d₆)3.82(s,3H),4.11(s,2H),6.86(dd,J＝7.6,1.2Hz,1H),6.94(dd,J＝7.8,1.2Hz,1H),7.01(t,J＝8.0Hz,1H),7.63(dd,J＝6.8,2.0Hz,2H),7.80(dd,J＝6.8,2.0Hz,2H),12.62(s,1H)；¹³C NMR(75MHz,DMSO-d₆)23.61,55.77,104.40,110.54,119.25,120.87,121.81,122.70,126.15(2C),129.53,131.76(3C),138.51,141.16,148.36; IR (Paraffin paste)

3435,1717,1639,1603,1576,1536,1463cm^-1；C₁₇H₁₃N₂O₂Br.1.9H₂Theoretical value of O: c, 52.12; h, 4.29; and N, 7.15. Experimental values: c, 52.12; h, 3.93; n, 7.37.

In one embodiment, 2- (2-fluorophenyl) -5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazole (MC 407). 2-fluorobenzaldehyde (0.0389 mg; 0.31mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) (0.0571 mg; 0.25mmol) in CH₃CN (2mL) and the suspension was stirred at 60 ℃ for 24 hours. Filtering the solid with CH₃CN was washed and identified as pure product. A beige solid; the yield is 82 percent; mp102-103 ℃;¹H NMR(400MHz,DMSO-d₆)3.82(s,3H),4.10(s,2H),6.86(dd,J＝7.6,2.4Hz,1H),6.94(dd,J＝8.0,2.4Hz,1H),7.00(t,J＝8.0Hz,1H),7.26-7.40(m,3H),7.95(td,J＝8.0,1.6Hz,1H),12.09(s,1H)；¹³CNMR(75MHz,DMSO-d₆)23.93,55.79,104.61,110.54,116.22(J ═ 16.1Hz),118.22(J ═ 8.6Hz),119.44,121.85,122.67,124.94(J ═ 2.3Hz),128.20(J ═ 2.2Hz),129.61(J ═ 6.2Hz),134.50,141.24,148.15,148.38,158.53(J ═ 184.5 Hz); IR (Paraffin paste) 3426,1710,1631,1603,1576,1536,1463cm^-1；C₁₇H₁₃N₂O₂F1.1H₂Theoretical value of O: c, 64.60; h, 4.46; and N, 8.87. Experimental values: c, 64.36; h, 4.46; and N, 8.91.

In one embodiment, 2- (3-fluorophenyl) -5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazole (MC 349). 3-fluorobenzaldehyde (0.0482 mg; 0.39mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) (0.0712 mg; 0.31mmol) in CH₃CN (1mL) and the suspension was stirred at 60 ℃ for 33 hours. Filtering the solid with CH₃CN was washed and identified as pure product. A beige solid; the yield is 100 percent; mp198-200 ℃;¹H NMR(400MHz,DMSO-d₆)3.82(s,3H),4.12(s,2H),6.86(dd,J＝7.6,1.2Hz,1H),6.94(dd,J＝8.0,1.2Hz,1H),7.00(t,J＝8.6Hz,1H),7.13(td,J＝8.6,2.8Hz,1H),7.44-7.51(m,1H),7.73(dt,J＝10.4,2.8Hz,1H),7.70(d,J＝7.6Hz,1H),12.58(s,1H)；¹³C NMR(75MHz,DMSO-d₆)23.60,55.79,104.46,110.56,110.68(J ═ 18.4Hz),114.43(J ═ 15.8Hz),119.24,120.23(J ═ 1.8Hz),121.81,122.68,130.95(J ═ 6.4Hz),132.78(J ═ 6.4Hz),138.39(J ═ 2.3Hz),141.21,148.31,148.38,162.52(J ═ 180.8 Hz); IR (Paraffin paste) 3356,1707,1652,1628,1522,1508,1461cm^-1；C₁₇H₁₃N₂O₂Theoretical value of F: c, 68.91; h, 4.42; and N, 9.45. Experimental values: c, 68.98; h, 4.58; and N, 9.66.

In one embodiment, 2- (4-chlorophenyl) -5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazole (MC 412). 4-Chlorobenzaldehyde (0.0291 mg; 0.21mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) (0.0417 mg; 0.18mmol) in CH₃In CN (1mL)To the suspension and the suspension is stirred at 60 ℃ for 24 hours. Filtering the solid with CH₃CN was washed and identified as pure product. A beige solid; the yield is 100 percent; mp212-214 ℃;¹H NMR(400MHz,DMSO-d₆)3.82(s,3H),4.12(s,2H),6.86(dd,J＝7.6,1.6Hz,1H),6.94(dd,J＝8.2,1.6Hz,1H),7.00(t,J＝8.0Hz,1H),7.50(d,J＝8.4Hz,2H),7.86(d,J＝8.4Hz,2H),12.53(s,1H)；¹³CNMR(75MHz,DMSO-d₆)23.62,55.76,104.18,110.52,119.25,121.80,122.62,125.84(2C),128.84(2C),129.36,132.18,138.55,141.22,148.31,148.36; IR (Paraffin paste) 3352,1700,1656,1601,1532,1489,1462cm^-1；C₁₇H₁₃N₂O₂Theoretical value of Cl: c, 65.29; h, 4.19; and N, 8.96. Experimental values: c, 65.47; h, 4.23; n, 8.74.

In one embodiment, 5-methoxy-2-phenyl-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazole (MC 350). Benzaldehyde (0.0520 mg; 0.49mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) (0.0653 mg; 0.29mmol) in CH₃CN (1mL) and the suspension was stirred at 60 ℃ for 33 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃The solid was washed with an aqueous solution of (0.05M), filtered and washed with water, resulting in the production of a pure product. A beige solid; the yield is 99 percent; mp 162-162 ℃;¹H NMR(400MHz,DMSO-d₆)3.83(s,3H),4.12(s,2H),6.86(dd,J＝7.6,1.6Hz,1H),6.94(dd,J＝8.0,1.6Hz,1H),7.00(t,J＝8.0Hz,1H),7.31(t,J＝7.6Hz,1H),7.43(t,J＝8.0Hz,2H),7.86(d,J＝7.8Hz,2H),12.43(s,1H)；¹³C NMR(75MHz,DMSO-d₆)23.68,55.77,110.50,119.33,121.83,122.56,124.20(2C),127.77,128.75(2C),130.50,139.62,141.29,148.23,148.38; IR (Paraffin paste)

3346,1702,1648,1602,1526,1496,1461cm^-1；C₁₇H₁₄N₂O₂Theoretical value of (2): c, 73.37; h, 5.07; n, 10.07. Experimental values: c, 73.58; h, 5.12; n, 10.34.

In one embodiment, 3- (5-methoxy) is synthesized-3, 9-dihydrobenzopyrano [2,3-d]Imidazol-2-yl) phenol (MC 359). 3-hydroxybenzaldehyde (0.0427 mg; 0.35mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-imido chloride (5) (0.0679 mg; 0.30mmol) in CH₃CN (1.2mL) and the suspension was stirred at 60 ℃ for 28 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃The solid was washed with an aqueous solution of (0.05M), filtered and washed with water, resulting in the production of a pure product. A pale green solid; the yield is 94%; mp 162-164 ℃;¹HNMR (400MHz, DMSO-d6) δ 3.82(s,3H),4.10(s,2H),6.71(dq, J ═ 8.2,1.2Hz,1H),6.85(dd, J ═ 7.8,1.2Hz,1H),6.92(dd, J ═ 8.2,1.2Hz,1H),7.00(t, J ═ 7.6Hz,1H),7.21(t, J ═ 7.6Hz,2H),7.27-7.30(m,2H),9.55(s,1H),12.33(s, 1H); 13C NMR (75MHz, DMSO-d6)23.70,55.75,110.47,111.23,114.99,115.07,119.36,121.84,122.54,129.74,131.77,139.76,141.30,148.10,148.37,157.62; IR (Paraffin paste) 3351,3218,1702,1659,1611,1523,1507,1460cm^-1(ii) a Theoretical value of C17H14N2O 3: c, 69.38; h, 4.79; n, 9.52. Experimental values: c, 69.42; h, 4.88; n, 9.56.

In one embodiment, 4- (5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazol-2-yl) phenol (MC 413). 4-hydroxybenzaldehyde (0.0388 mg; 0.32mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) (0.0579 mg; 0.26mmol) in CH₃CN (2mL) and the suspension was stirred at 60 ℃ for 48 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃The solid was washed with an aqueous solution of (0.05M), filtered and washed with water, resulting in the production of a pure product. A yellow solid; the yield is 64 percent; mp 245-246 ℃;¹HNMR(400MHz,DMSO-d6)3.82(s,3H),4.09(s,2H),6.81(dd,J＝6.8,2.0Hz,2H),6.85(dd,J＝7.6,1.6Hz,1H),6.92(dd,J＝8.0,1.6Hz,1H),6.99(t,J＝8.0Hz,1H),7.68(dd,J＝6.8,2.0Hz,2H),9.63(s,1H),12.11(s,1H)；13C NMR(75MHz,DMSO-d6)23.76,55.75,102.26,110.43,115.48(2C),119.45,121.86(2C),122.45,125.88(2C),140.34,141.36,147.86,14837,157.44; IR (Paraffin paste) 3338,3246,1706,1645,1612,1548,1503,1463cm^-1(ii) a Theoretical value of C17H14N2O 3: c, 69.38; h, 4.79; n, 9.52. Experimental values: c, 69.39; h, 4.42; n, 9.54.

In one embodiment, 5-methoxy-2- (2-methoxyphenyl) -3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazole (MC 411). 2-Methoxybenzaldehyde (0.0632 mg; 0.46mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-imido chloride (5) (0.0780 mg; 0.34mmol) in CH₃CN (1mL) and the suspension was stirred at 60 ℃ for 47 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃The solid was washed with an aqueous solution of (0.05M), filtered and washed with water, resulting in the production of a pure product. A yellow solid; the yield is 85 percent; mp188-190 deg.C;¹H NMR(400MHz,DMSO-d₆)3.82(s,3H),3.95(s,3H),4.10(s,2H),6.86(dd,J＝7.6,1.6Hz,1H),6.93(dd,J＝8.0,1.6Hz,1H),6.97-7.04(m,2H),7.13(dd,J＝8.0,0.8Hz,1H),7.30(td,J＝7.7,2.0Hz,1H),7.99(dd,J＝8.0,2.0Hz,1H),11.63(s,1H)；¹³C NMR(100MHz,DMSO-d₆)

24.30,55.50,55.78,103.05,110.47,111.72,118.44,119.67,120.78,121.91,122.51,127.39,128.95,137.14,141.34,147.76,148.38,155.59; IR (Paraffin paste)

3340,1706,1659,1594,1530,1506,1460cm^-1；C₁₈H₁₆N₂O₃Theoretical value of (2): c, 70.12; h, 5.23; and N, 9.09. Experimental values: c, 70.44; h, 5.20; and N, 9.16.

In one embodiment, 2, 4-difluoro-6- (5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazol-2-yl) phenol (MC 410). 2, 4-difluoro-6-hydroxybenzaldehyde (0.0493 mg; 0.31mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-imido chloride (5) (0.061 mg; 0.27mmol) in CH₃CN (2mL) and the suspension was stirred at 60 ℃ for 36 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃(0.05M) in waterThe solid was washed with water, filtered and washed with water, resulting in the production of a pure product. A yellow solid; the yield is 49 percent; mp260-262 ℃;¹H NMR(400MHz,DMSO-d₆)3.83(s,3H),4.15(s,2H),6.87(dd,J＝7.6,2.4Hz,1H),6.96(dd,J＝8.2,2.4Hz,1H),7.04(t,J＝8.0Hz,1H),7.24(td,J＝10.0,1.6Hz,1H),7.51(dt,J＝10.0,1.6Hz,1H),11.91(s,1H),12.90(s,1H)；¹³C NMR(75MHz,DMSO-d₆)23.28,55.70,104.31(dd, J ═ 20.6,16.5Hz),104.39,105.38(dd, J ═ 18.8,4.5Hz),110.59,115.36(dd, J ═ 7.9,4.5Hz),119.03,121.71,123.14,137.71-137.79(m),140.30(J ═ 10.2,4.5Hz),140.68,145.62,148.29,150.80(dd, J ═ 181.9,10.0),153.9(dd, J ═ 175.9, 8.8); IR (Paraffin paste) 3348,3225,1698,1651,1601,1542,1461cm^-1；C₁₇H₁₂N₂O₃F₂0.8H₂Theoretical value of O: c, 59.23; h, 3.72; and N, 8.13. Experimental values: c, 59.13; h, 3.53; and N, 8.42.

In one embodiment, 2- (4-ethoxyphenyl) -5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazole (MC 415). 4-ethoxybenzaldehyde (0.0403 mg; 0.30mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-iminium chloride (5) (0.0614 mg; 0.27mmol) in CH₃CN (2mL) and the suspension was stirred at 60 ℃ for 47 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃The solid was washed with an aqueous solution of (0.05M), filtered and washed with water, resulting in the production of a pure product. A yellow solid; the yield is 96 percent; mp215-217 ℃;¹H NMR(400MHz,DMSO-d₆)1.20(t,J＝7.6Hz,3H),2.62(q,J＝7.6Hz,2H),3.84(s,3H),4.12(s,2H),6.87(dd,J＝7.2,1.6Hz,1H),6.94(dd,J＝8.2,1.2Hz,1H),7.01(t,J＝7.6Hz,1H),7.28(d,J＝8.4Hz,2H),7.79(dd,J＝6.6,1.6Hz,2H),12.36(s,1H)；¹³C NMR(75MHz,DMSO-d6)

15.43,23.73,27.94,55.80,103.30,110.52,119.41,121.87,122.59,124.33(2C),128.08,128.16(2C),139.87,141.33,143.60,148.40; IR (Paraffin paste)

3342,1703,1656,1610,1539,1502,1460cm^-1；C₁₉H₁₈N₂O₃Theoretical value of (2): c, 70.81; h, 5.59; and N, 8.70. Experimental values: c, 70.74; h, 5.67; n, 8.77.

In one embodiment, 2-bromo-6-methoxy-3- (5-methoxy-3, 9-dihydrobenzopyrano [2,3-d ] is synthesized]Imidazol-2-yl) phenol (MC 416). 2-bromo-3-hydroxy-4-methoxybenzaldehyde (0.0780 mg; 0.34mmol) was added to 3-amino-8-methoxy-2H-benzopyran-2-imido chloride (5) (0.0607 mg; 0.27mmol) in CH₃CN (1mL) and the suspension was stirred at 60 ℃ for 46 hours. Filtering the solid with CH₃CN was washed and identified as pure product. When in¹When HCl contamination is observed in the H NMR spectrum, NaHCO is used₃The solid was washed with an aqueous solution of (0.05M), filtered and washed with water, resulting in the production of a pure product. A beige solid; the yield is 71 percent; mp 160-161 ℃;¹H NMR(400MHz,DMSO-d₆)3.82(s,3H),3.86(s,3H),4.08(s,2H),6.85(dd,J＝7.6,1.2Hz,1H),6.93(dd,J＝8.0,1.6Hz,1H),6.99(t,J＝8.0Hz,1H),7.03-7.08(m,2H),9.55(s,1H),11.97(s,1H)；¹³C NMR(75MHz,DMSO-d₆)23.82,55.74,56.23,102.75,109.30,110.43,110.54,119.46,121.38,121.84,122.46,125.26,139.04,141.33,144.05,147.52,148.29,148.37; IR (Paraffin paste) 3338,3212,1703,1647,1535,1497,1461cm^-1；C₁₈H₁₅N₂O₄Br1.8H₂Theoretical value of O: c, 49.61; h, 3.86; n, 6.43. Experimental values: c, 49.68; h, 3.91; n, 6.43.

In one embodiment, 5' -dimethoxy-2, 2' -diphenyl-1, 1',9,9' -tetrahydro-9, 9' -dibenzopyrano [2,3-d ] is synthesized]Imidazole. Aldehyde (1) (1-1.2 equiv.) is added to 2-imino-8-methoxy-2H-benzopyran-3-amine (4) in CH₃CN (1-2mL) and the solution was stirred at 80 ℃ for 7-24 hours. The solid product began to precipitate slowly, was filtered and washed with CH₃CN was washed and identified as pure product 8.

In one embodiment, 2' -bis (4-fluorophenyl) -5,5' -dimethoxy-1, 1',9,9' -tetrahydro-9, 9' -dibenzopyrano [2,3-d ] is synthesized]Imidazole (MC 406). 4-fluorobenzaldehyde (0.0629 mg; 0.51mmol) was added to 2-imino-8-methoxylbenzaldehydeYl-2H-benzopyran-3-amine (4) (0.0962 mg; 0.51mmol) in CH₃CN (1mL) and the solution was stirred at 80 ℃ for 7 hours. The solid product began to precipitate slowly, was filtered and washed with CH₃CN was washed and identified as pure product. A beige solid; the yield is 18 percent; mp 272 and 274 ℃;¹H NMR(400MHz,DMSO-d₆)3.68(s,6H),4.89(s,2H),5.80(dd,J＝7.8,2.0Hz,2H),6.71(t,J＝7.8Hz,2H),6.80(dd,J＝8.7,1.6Hz,2H),7.32-7.49(m,4H),7.98-8.03(m,4H),12.57(s,2H)；¹³C NMR(75MHz,DMSO-d₆)41.21,55.64,105.81,111.00,115.83(2C, J ═ 21.2),119.62,120.44,122.01,126.80(2C, J ═ 8.0),127.12,139.98,141.90,147.62,150.12,162.00(J ═ 243.8); IR (Paraffin paste) 3348,1700,1650,1608,1538,1500,1461cm^-1；C₃₄H₂₄N₄O₄F₂1.2H₂Theoretical value of O: c, 64.23; h, 4.85; and N, 8.82. Experimental values: c, 64.21; h, 4.79; and N, 8.80.

In one embodiment, 2' -bis (4-bromophenyl) -5,5' -dimethoxy-1, 1',9,9' -tetrahydro-9, 9' -dibenzopyrano [2,3-d ] is synthesized]Imidazole (MC 421). 4-Bromobenzaldehyde (0.069 mg; 0.37mmol) was added to 2-imino-8-methoxy-2H-benzopyran-3-amine (4) (0.0705 mg; 0.37mmol) in CH₃CN (1.5mL) and the solution was stirred at 80 ℃ for 8 hours. The solid product began to precipitate slowly, was filtered and washed with CH₃CN was washed and identified as pure product. A beige solid; the yield is 32%; mp 266-268 ℃;¹H NMR(400MHz,DMSO-d₆)3.68(s,6H),4.89(s,2H),5.78(dd,J＝7.8,1.6Hz,2H),6.71(t,J＝7.8Hz,2H),6.80(dd,J＝8.2,2.0Hz,2H),7.71(dd,J＝6.6,1.4Hz,2H),7.91(dd,J＝6.9,1.6Hz,2H),12.69(s,2H)；¹³C NMR(75MHz,DMSO-d₆)41.24,55.65,106.33,111.06,119.52,120.41,121.14,122.06,126.58,129.65,131.80,139.70,141.86,147.62,150.25; IR (Paraffin paste) 3352,1703,1654,1610,1543,1508,1461cm^-1；C₃₄H₂₄N₄O₄Br₂Theoretical value of (2): c, 57.32; h, 3.40; and N, 7.86. Experimental values: c, 57.78; h, 3.37; and N, 7.88.

In one embodiment, 2' -bis (2-fluorophenyl) -5,5' is synthesized '-dimethoxy-1, 1',9,9' -tetrahydro-9, 9' -dibenzopyrano [2,3-d ]]Imidazole (MC 369). 2-fluorobenzaldehyde (0.0446 mg; 0.36mmol) was added to 2-imino-8-methoxy-2H-chromen-3-amine (4) (0.0590 mg; 0.31mmol) in CH₃CN (2mL) and the solution was stirred at 80 ℃ for 24 hours. The solid product began to precipitate slowly, was filtered and washed with CH₃CN was washed and identified as pure product. A beige solid; the yield is 15 percent; mp 276-278 ℃;¹H NMR(400MHz,DMSO-d₆)3.69(s,6H),5.00(s,2H),5.91(dd,J＝8.0,1.2Hz,2H),6.75(t,J＝8.0Hz,2H),6.82(dd,J＝8.2,1.6Hz,2H),7.31-7.48(m,6H),7.99(td,J＝5.7,1.6Hz,2H),11.88(s,2H)；¹³C NMR(75MHz,DMSO-d₆)41.08,55.65,106.63,110.99,116.28(J ═ 21.3),118.24(J ═ 11.4),120.02,120.45,122.06,124.98,128.60(J ═ 2.8),129.98(J ═ 8.2),135.58,141.90,147.62,149.93,158.73(J ═ 246.6); IR (Paraffin paste) 3438,3447,1637,1575,1614,1575,1530,1469cm^-1；C₃₄H₂₄N₄O₄F₂.0.8H₂Theoretical value of O: c, 67.51; h, 4.23; and N, 9.26. Experimental values: c, 67.58; h, 4.30; and N, 9.11.

Whenever used in this document, the term "comprising" is intended to specify the presence of stated features, integers, steps, components, but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

Those skilled in the art will appreciate that the particular order of the steps described is illustrative only and can be varied without departing from the invention unless otherwise indicated herein. Accordingly, unless otherwise specified, the steps described are unordered meaning that, when possible, the steps can be performed in any convenient or desirable order.

Where the singular form of an element or feature is used in the specification or claims, the plural is also included if not specifically excluded. For example, the term "a compound" or "the compound" also includes the plural form "a plurality of compounds" or "the plurality of compounds" and vice versa. In the claims, articles such as "a" and "the" may mean one or more than one unless specified to the contrary or otherwise apparent from the context. Claims or descriptions that include an "or" between one or more members of a group are deemed to be satisfied if present, used, or otherwise relevant to one, more than one, or all of the group members in a given product or process unless stated to the contrary or otherwise apparent from the context. The present invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or process. The invention also includes embodiments in which more than one, or all, of the group members are present in, used in, or otherwise relevant to a given product or process.

Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more claims or from the relevant portion of the description is introduced into another claim. For example, any claim that depends from another claim may be altered to include one or more limitations that exist in any other claim that depends from the same base claim.

Further, when the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are to be included, and methods of making the composition according to the methods of making disclosed herein or other methods known in the art are to be included, unless otherwise indicated or unless it is apparent to one of ordinary skill in the art that a contradiction or inconsistency would arise.

When ranges are given, endpoints are included. Further, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or understanding of one of ordinary skill in the art, values expressed as ranges can assume any specific value within the ranges recited in the various embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is further understood that unless otherwise indicated or otherwise evident from the context and/or understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

The invention should not be considered in any way as being limited to the described embodiments and many possibilities to modifications thereof will be foreseen by a person skilled in the art.

The above embodiments are combinable.

Reference to the literature

(1)https://www.uicc.org/new-global-cancer-data-globocan-2018(02/01/2019)

(2)http://gco.iarc.fr(02/01/2019)

(3)Bao,B；Mitrea,C.；Wijesinghe,P.；Marchetti,L.；Girsch,E.；Farr,R.；Boerner,J.；Mohammad,R.；Dyson,G.；Terlecky,S.and Bollig-Fischer,A.Treating triple negative breast cancer cells with erlotinib plus a select antioxidant overcomes drug resistance by targeting cancer cell heterogeneity.Scientific Reports 2017,7,44125.

(4)Costa,M.；Dias,T.；Brito,A.And

F.Biological importance of structurally diversified chromenes.Eur.J.Med.Chem,2016,123,487-507.

(5)Costa,M.；Rodrigues,A.I.；Proenca,F.Synthesis of3-aminochromenes:the Zincke reaction revisited.Tetrahedron 2014,70(33),4869.

(6)Costa,M.；Proenca,F.2-Aryl-1,9-dihydrochromeno[3,2-d]imidazoles:a facile synthesis fromsalicylaldehydes and arylideneaminoacetonitrile.Tetrahedron 2011,67(10),1799。

Claims

1. A compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph for use in medicine or veterinary comprising the formula:

wherein

R¹、R²And R³Selecting independently of each other;

R¹selected from aryl or heterocycle;

R²selected from H, alkyl, aryl, alkoxy, halogen, hydroxy, amine, carbonyl, or heterocycle;

R³is selected from H or

Provided that it does not comprise

2. A compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph according to preceding claim, wherein

R¹Is aryl;

R²selected from H, alkyl, alkoxy, halogen, hydroxy or amine.

3. The compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph according to the preceding claim, wherein the dimer is preferably a homodimer.

4. The compound or pharmaceutically acceptable salt of any one of the preceding claimsA hydrate, solvate, N-oxide, stereoisomer, diastereoisomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein R¹Is a substituted aryl group, preferably a substituted phenyl group.

5. The compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph of any preceding claim, wherein R is¹Selected from the following list: hydroxyphenyl, hydroxy-methoxyphenyl, hydroxy-bromophenyl, hydroxy-chlorophenyl, fluorophenyl, bromophenyl, 24-chlorophenyl, phenyl, methoxyphenyl, difluoro-hydroxyphenyl, ethoxyphenyl or bromo-hydroxy-methoxyphenyl.

6. The compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph of any preceding claim, wherein R is¹Is 2-hydroxyphenyl, 2-hydroxy-3-methoxyphenyl, 2-hydroxy-5-bromophenyl, 2-hydroxy-5-chlorophenyl, 4-fluorophenyl, 4-bromophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-chlorophenyl, phenyl, 3-hydroxyphenyl, 2-methoxyphenyl, 3, 5-difluoro-2-hydroxyphenyl, 4-ethoxyphenyl or 2-bromo-3-hydroxy-4-methoxyphenyl.

7. The compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph of any preceding claim, wherein R is²Is a substituted or unsubstituted aryl group.

8. The compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, or pharmaceutically acceptable salt of any of the preceding claimsAn enantiomer, atropisomer, dimer or polymorph wherein R²Selected from the following list: H. 5-methoxy, 7-bromo, 7-chloro or 7-fluoro.

9. The compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph of any preceding claim, wherein R is³Is a benzopyran unit or H.

10. The compound or pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph of any preceding claim, wherein R is³Selected from the following list: H. 2- (4-fluorophenyl) -5-methoxy-1, 9-dihydrobenzopyrano [2,3-d]Imidazole unit, 2- (4-bromophenyl) -5-methoxy-1, 9-dihydrobenzopyrano [2,3-d]Imidazole unit or 2- (2-fluorophenyl) -5-methoxy-1, 9-dihydrobenzopyrano [2,3-d]An imidazole unit.

11. The compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate, solvate, N-oxide, stereoisomer, diastereomer, enantiomer, atropisomer, dimer or polymorph thereof, wherein the compound is selected from the following list:

12. a compound according to any one of the preceding claims 2-11 for use in medicine or veterinary medicine.

13. A compound according to any preceding claim for use in the treatment, therapy or diagnosis of a disease characterised by benign or malignant cell proliferation or by areas of neovascularization or excessive vascularization or cancer.

14. A compound according to any preceding claim for use in the treatment, therapy or diagnosis of hyperproliferative tissue or neoplasia.

15. A compound according to any preceding claim for use in the treatment, therapy or diagnosis of breast cancer, renal cell carcinoma, leukaemia, glioma or glioblastoma.

16. A compound according to any preceding claim for use in the treatment, therapy or diagnosis of renal cell carcinoma, leukemia, glioma, glioblastoma, breast cancer.

17. A compound according to any preceding claim for use in the treatment, therapy or diagnosis of triple negative breast cancer, acute renal cell carcinoma, luminal breast cancer, basal-like breast cancer, acute leukemia.

18. A pharmaceutical composition comprising at least one of the compounds according to any one of the preceding claims.

19. The pharmaceutical composition according to the preceding claim, further comprising a pharmaceutically acceptable carrier.

20. The pharmaceutical composition of any one of the preceding claims 19-20, further comprising an antiviral agent, an analgesic agent, an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic agent, an antifungal agent, an antiparasitic agent, an antibiotic, a diuretic, or a mixture thereof.

21. The pharmaceutical composition of any one of the preceding claims 19-21, further comprising a filler, binder, disintegrant, or lubricant, or a mixture thereof.

22. The pharmaceutical composition according to any of the preceding claims 19-22 for intradermal, topical, systemic, transdermal, intravenous therapy or combinations thereof.

23. A nanoparticle comprising a compound of claims 1-18 and/or a pharmaceutical composition of claims 19-23.

24. The nanoparticle of the preceding claim, wherein the compound is encapsulated by the nanoparticle.

25. A kit comprising a compound according to claims 1-18 and/or a pharmaceutical composition according to claims 19-23.